Method and apparatus for aligning microbeads in order to interrogate the same

ABSTRACT

A method and apparatus are provided for aligning optical elements or microbeads  8 , wherein each microbead has an elongated body with a code embedded therein along a longitudinal axis thereof to be read by a code reading device. The microbeads  8  are aligned with a positioning device (or cell)  500  having a plate or platform  200, 1252  with grooves  205, 1258  so the longitudinal axis of the microbeads is positioned in a fixed orientation relative to the code reading device. The microbeads  8  are typically cylindrically shaped glass beads having a diffraction grating-based code embedded in the bead  8  disposed along an axis, which requires a predetermined alignment between the incident code readout laser beam and the code readout detector in two of three rotational axes. The geometry of the grooves  205  are designed to allow for easy loading and unloading of beads from a cell, and the grooves  205  may be straight or curved. Also, the cell may be segmented into regions each associated with a different reaction or used for a different identification process/application, and may have many different geometries depending on the application.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of: U.S. ProvisionalApplication Ser. No. 60/609,583, filed Sep. 13, 2004, entitled “ImprovedMethod and Apparatus for Aligning Microbeads in Order to InterrogateSame” (Docket No. CV-0082 PR); Ser. No. 60/610,910, filed Sep. 17, 2004,entitled “Method and Apparatus for Aligning Microbeads in Order toInterrogate Same” (Docket No. CV-0086 PR); and Ser. No. 60/610,833,filed Sep. 17, 2004, entitled “Method and Apparatus for Transporting andKitting Microbeads” (Docket No. CV-0087 PR); and is acontinuation-in-part of: U.S. patent application Ser. No. 10/661,836,filed Sep. 12, 2003, entitled “Method And Apparatus For AligningMicrobeads In Order To Interrogate The Same” (Docket No. CV-0042), andSer. No. 11/063,665, filed Feb. 22, 2005, entitled “Multi-well Platewith Alignment Grooves for Encoded Microparticles” (Docket No. CV-0053US), all the above of which are incorporated herein by reference intheir entirety.

The following cases contain subject matter related to that disclosedherein and are all incorporated herein by reference in their entirety:U.S. patent application Ser. No. 10/661,234, filed Sep. 12, 2003,entitled “Diffraction Grating-Based Optical Identification Element”,(Docket No. CV-0038A); Ser. No. 10/661,031, filed Sep. 12, 2003,entitled “Diffraction Grating-Based Encoded Micro-particles forMultiplexed Experiments”, (Docket No. CV-0039A); Ser. No. 10/661,082,filed Sep. 12, 2003, entitled “Method and Apparatus for Labeling UsingDiffraction Grating based Encoded Optical Identification Elements”(Docket No. CV-0040); U.S. patent application Ser. No. 10/661,115, filedSep. 12, 2003, entitled “Assay Stick” (Docket No. CV-0041); Ser. No.10/661,254 filed Sep. 12, 2003, entitled “Chemical Synthesis UsingDiffraction Grating-Based Encoded Optical Elements” (Docket No.CV-0043); U.S. patent application Ser. No. 10/661,116, filed Sep. 12,2003, entitled “Method Of Manufacturing Of A Diffraction Grating-BasedIdentification Element” (Docket No. CV-0044); and U.S. patentapplication Ser. No. 10/763,995, filed Jan. 22, 2004, entitled, “HybridRandom Bead/Chip Based Microarray” (Docket No. CV-0054); and U.S. patentapplication Ser. No. 10/956,791, filed Oct. 1, 2004, entitled “OpticalReader for Diffraction Grating-Based Encoded Optical IdentificationElements” (Docket No. CV-0092 US).

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention generally relates to a method and apparatus forprocessing information contained on microbeads, each microbead having anelongated body with a code embedded therein along a longitudinal axisthereof to be read by a code reading device; and more particularly to amethod and apparatus for aligning the microbeads so the longitudinalaxis thereof is in a fixed orientation relative to the code reading orother device.

This invention also relates to transporting beads, and more particularlyto transporting microbeads from one location to another.

2. Description of Related Art

Many industries have a need for uniquely identifiable objects or for theability to uniquely identify objects, for sorting, tracking, and/oridentification/tagging. Existing technologies, such as bar codes,electronic microchips/transponders, radio-frequency identification(RFID), and fluorescence and other optical techniques, are ofteninadequate. For example, existing technologies may be too large forcertain applications, may not provide enough different codes, or cannotwithstand harsh temperature, chemical, nuclear and/or electromagneticenvironments.

Therefore, it would be desirable to obtain a coding element or platformthat provides the capability of providing many codes (e.g., greater than1 million codes), that can be made very small and/or that can withstandharsh environments.

Moreover, it would be desirable to provide a method and apparatus toposition and align such encoded elements so as to identify the code todetermine information about the process or application to which it isrelated and/or to better sense the chemical content on the elements andcorrelate it in relation to such process or application.

It is also well known that microbeads or microparticles may be used forvarious types of multiplexed chemical experiments or assays or foridentifying, authenticating or sorting items. One challenge intransporting microbeads is being able to move them reliably from onelocation to another reliably and/or being able to move a predeterminednumber of beads.

Accordingly, it would be desirable to provide a reliable technique fortransporting microbeads from one location to another.

SUMMARY OF THE INVENTION

In its broadest sense, the present invention provides a new and uniquemethod and apparatus for aligning new and unique coding elements ormicrobeads, wherein each microbead has an elongated body with a codeembedded therein along a longitudinal axis thereof to be read by a codereading or other detection device. The method features the step ofaligning the microbeads with a positioning device so the longitudinalaxis of the microbeads is positioned in a fixed orientation relative tothe code reading or other detection device.

The new and unique microbeads are not spherical, but instead have anelongated shape and may be cylindrical, cubic, rectangular, or any otherelongated shape. The microbeads are typically composed of silica glasswith some germanium and/or boron doped region or regions that arephotosensitive to ultraviolet light. Coded microbeads are individuallyidentifiable via a single or series of spatially overlapping pitcheswritten into them. The microbeads may be used in many differentprocesses. After such processing, the microbeads have a resultingchemical content on the surface of each bead that is sensed andcorrelated in relation to the code contained with the microbead todetermine information about the process.

When used in an assay process, the microbeads are typicallycylindrically (i.e. tubular) shaped glass beads and between 25 and 250μm in diameter and between 100 and 500 μm long. Other sizes may be usedif desired. They have a holographic code embedded in the central regionof the bead, which is used to identify it from the rest of the beads ina batch of beads with many different DNA or other chemical probes. Across reference is used to determine which probe is attached to whichbead, thus allowing the researcher to correlate the chemical content oneach bead with the measured fluorescence signal. Because the codeconsists of a diffraction grating 12 typically disposed along an axis ofthe microbead, there is a particular alignment required between theincident readout laser beam and the readout detector in two of the threerotational axes. In aeronautical terms, the two of the three rotationalaxes include the pitch of the microbead in the front-to-back directionand the yaw of the microbead in a side-to-side direction. The thirdaxis, rotation about the center axis of the cylinder, is azimuthallysymmetric and therefore does not require alignment. The third axis isanalogous to the roll axis.

The invention provides a method for aligning the microbeads in the tworotational axes to a fixed orientation relative to an incident laserbeam and a readout camera, otherwise known as the code camera. Theinvention further provides a method for rapidly aligning a large numberof microbeads, between 1,000 and 1,000,000 microbeads or more,economically, and with the necessary tolerances. The method is flexibleas it relates to the size of the microbeads and can be integrated into afully automated system, which prepares the microbeads for rapid readoutby an automated code-reading machine.

In one embodiment of the present invention, the positioning deviceincludes a plate with a series of parallel grooves (or channels), whichcould have one of several different shapes, including square,rectangular, v-shaped, semi-circular, etc., as well as a flat bottomgroove with tapered walls. The grooves are formed into an opticallytransparent medium such as boro-silicate glass, fused silica, or otherglasses, or plastic or other transparent support materials. The depth ofthe grooves will depend on the diameter of the microbead but generallythey will be between 10 and 125 μm, but may be larger as discussedhereinafter, depending on the application. The spacing of the grooves ismost optimal when it is between 1 and 2 times the diameter of themicrobead, providing for both maximum packing density as well as maximumprobability that a microbead will fall into a groove. The width ofgrooves is most optimal when the gap between the microbead and the wallsof the grooves is sufficiently small to prevent the microbeads fromrotating within the grooves by more than a few degrees. The bottom ofthe groove must also be maintained flat enough to prevent the microbeadsfrom rotating, by more than a few tenths of a degree, relative to theincident laser beam. Another critical aspect of the grooved plate is theoptical quality of the grooves. To prevent excess scatter of the readoutlaser beam, which could lead to low contrast between the code signal andthe background scatter, it is important that the grooves exhibit highoptical quality. The beads can be read in the groove plate from below,on top of, or the side of the plate, depending on the application andtype of microbead used.

Some advantages of the groove plate approach include:

Rapid simultaneous alignment of microbeads. Alignment rates ˜1000's persecond.

Once the microbeads are aligned, they can be read as many times asnecessary to get a good reading or improve statistics.

Microbeads naturally fall into groove (presumably by capillary forces)at very high packing densities.

Microbeads can be mixed after reading then re-read to enhance thestatistics of readout process.

In an alternative embodiment of the present invention, the positioningdevice may includes a tube having a bore for receiving, aligning andreading the microbeads.

Moreover, the present invention also provides an apparatus for aligningan optical identification element. The optical identification elementhaving an optical substrate having at least a portion thereof with atleast one diffraction grating disposed therein, the grating having atleast one refractive index pitch superimposed at a common location, thegrating providing an output optical signal when illuminated by anincident light signal, the optical output signal being indicative of acode, and the optical identification element being an elongated objectwith a longitudinal axis. The apparatus also having an alignment devicewhich aligns the optical identification element such that said outputoptical signal is indicative of the code.

The present invention also provides an optical element capable of havingmany optically readable codes. The element has a substrate containing anoptically readable composite diffraction grating having one or morecollocated index spacing or pitches Λ. The invention allows for a highnumber of uniquely identifiable codes (e.g., millions, billions, ormore). The codes may be digital binary codes and thus are digitallyreadable or may be other numerical bases if desired.

Also, the elements may be very small “microbeads” (or microelements ormicroparticles or encoded particles) for small applications (about1-1000 microns), or larger “macroelements” for larger applications(e.g., 1-1000 mm or much larger). The elements may also be referred toas encoded particles or encoded threads. Also, the element may beembedded within or part of a larger substrate or object.

The code in the element is interrogated using free-space optics and canbe made alignment insensitive.

The gratings (or codes) are embedded inside (including on or near thesurface) of the substrate and may be permanent non-removable codes thatcan operate in harsh environments (chemical, temperature, nuclear,electromagnetic, etc.).

The code is not affected by spot imperfections, scratches, cracks orbreaks in the substrate. In addition, the codes are spatially invariant.Thus, splitting or slicing an element axially produces more elementswith the same code. Accordingly, when a bead is axially split-up, thecode is not lost, but instead replicated in each piece.

The invention is a significant improvement over prior art bead movementtechniques in being able to repeatably move a predetermined number ofbeads from one location (or container or well) to another location (orcontainer or well). Also, the invention provides for the reliable andrepeatable transportation of all beads from one container or well toanother or from one well to multiple wells using a “telegraph”technique. The invention is useful for creating multiplexed bead kitshaving a required number of beads of each code in a kit. The presentinvention may also be used to move the beads from a container to areader to allow for the bead codes and/or chemistry on the beads to beread. The invention may be used in any assay or multiplexed experiment,combinatorial chemistry or biochemistry assay process, or in a taggantapplication, or any other application where beads are in a liquidsolution and need to be transported, kitted and/or read.

Advantages of the “telegraph” technique of the present invention arethat it is low cost, fast, effective/reliable for moving beads, and lowprecision is required. Advantages of the pipetting techniques of thepresent invention is that the pippeter is a standard off the shelfproduct, it is flexible to be used with any type of well or container(e.g., sizes, shapes and other characteristics), or other fluidconfigurations, and does not require any sealing or physical connectionsto the wells.

The foregoing and other objects, features and advantages of the presentinvention will become more apparent in light of the following detaileddescription of exemplary embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWING

The drawing is not drawn to scale and includes the following Figures:

FIG. 1 shows the steps of a microbead platform assay process.

FIG. 2 is a side view of an optical identification element, inaccordance with the present invention.

FIG. 3 is a top level optical schematic for reading a code in an opticalidentification element, in accordance with the present invention.

FIG. 4 is a perspective view of a grooved plate for use with an opticalidentification element, in accordance with the present invention.

FIG. 5 is a diagram of the flat grooves and an example of thedimensionality thereof in accordance with the present invention.

FIG. 6 is a perspective view of a plate with holes for use with anoptical identification element, in accordance with the presentinvention.

FIG. 7 is a perspective view of a grooved plate for use with an opticalidentification element, in accordance with the present invention.

FIG. 8 is a diagram of a microbead mapper reading, in accordance withthe present invention.

FIG. 8 a is a diagram of a system for both detecting a material on andreading a code in a microbead, in accordance with the present invention.

FIG. 9 is a diagram of a plate having microbeads thereon in relation toan open plate format for detection and reading of the microbead inaccordance with the invention.

FIG. 10 is a diagram of a starting point for handling microbeads forreadout in a cuvette process in accordance with the invention.

FIG. 11 is a diagram of a second step in the readout process inaccordance with the invention.

FIG. 12 is a diagram of the readout step in accordance with theinvention.

FIG. 13 is a diagram of final steps in the cuvette process in accordancewith the invention.

FIG. 14 is a diagram of an example of the cuvette showing its mount on akinematic plate in accordance with the invention.

FIG. 15 is a diagram of an alternative embodiment of a cuvette showing aport for fluid filling/emptying using a pipette in accordance with theinvention.

FIG. 16 is a diagram of an alternative embodiment of a cuvette showingan alternative port for fluid filling/emptying using a pipette inaccordance with the invention.

FIG. 17 is a diagram of a two zone cuvette showing a free region and atrapped region in accordance with the invention.

FIG. 18(a) is a diagram of steps for a conventional flow cytometerreader in a single pass cytometer process in accordance with theinvention.

FIG. 18(b) is a diagram of steps for a disk cytometer reader in amultipass cytometer process in accordance with the invention.

FIGS. 19(a), (b) and (c) show embodiments of a disk cytometer inaccordance with the invention.

FIG. 20(a) show an embodiment of a disk cytometer having radial channelsfor spin drying in accordance with the invention.

FIG. 20(b) show an alternative embodiment of a disk cytometer having amechanical iris for providing a variable aperture for bead access togrooves in accordance with the invention.

FIG. 21 show an embodiment of a SU8 groove plate having 450 inaccordance with the invention.

FIG. 21 show an embodiment of a SU8 cylindrical grooved plate having450×65 microns beads in accordance with the invention.

FIG. 22 show an embodiment of an alignment tube in accordance with theinvention.

FIG. 23 show an alternative embodiment of an alignment tube having areceiving flange in accordance with the invention.

FIG. 24 is an optical schematic for reading a code in an opticalidentification element, in accordance with the present invention.

FIG. 25(a) is an image of a code on a CCD camera from an opticalidentification element, in accordance with the present invention.

FIG. 25(b) is a graph showing an digital representation of bits in acode in an optical identification element, in accordance with thepresent invention.

FIG. 26 illustrations (a)-(c) show images of digital codes on a CCDcamera, in accordance with the present invention.

FIG. 27 illustrations (a)-(d) show graphs of different refractive indexpitches and a summation graph, in accordance with the present invention.

FIG. 28 is an alternative optical schematic for reading a code in anoptical identification element, in accordance with the presentinvention.

FIG. 29 illustrations (a)-(b) are graphs of reflection and transmissionwavelength spectrum for an optical identification element, in accordancewith the present invention.

FIGS. 30-31 are side views of a thin grating for an opticalidentification element, in accordance with the present invention.

FIG. 32 is a perspective view showing azimuthal multiplexing of a thingrating for an optical identification element, in accordance with thepresent invention.

FIG. 33 is side view of a blazed grating for an optical identificationelement, in accordance with the present invention.

FIG. 34 is a graph of a plurality of states for each bit in a code foran optical identification element, in accordance with the presentinvention.

FIG. 35 is a side view of an optical identification element where lightis incident on an end face, in accordance with the present invention.

FIGS. 36-37 are side views of an optical identification element wherelight is incident on an end face, in accordance with the presentinvention.

FIG. 38, illustrations (a)-(c), are side views of an opticalidentification element having a blazed grating, in accordance with thepresent invention.

FIG. 39 is a side view of an optical identification element having acoating, in accordance with the present invention.

FIG. 40 is a side view of whole and partitioned optical identificationelement, in accordance with the present invention.

FIG. 41 is a side view of an optical identification element having agrating across an entire dimension, in accordance with the presentinvention.

FIG. 42, illustrations (a)-(c), are perspective views of alternativeembodiments for an optical identification element, in accordance withthe present invention.

FIG. 43, illustrations (a)-(b), are perspective views of an opticalidentification element having multiple grating locations, in accordancewith the present invention.

FIG. 44, is a perspective view of an alternative embodiment for anoptical identification element, in accordance with the presentinvention.

FIG. 45 is a view an optical identification element having a pluralityof gratings located rotationally around the optical identificationelement, in accordance with the present invention.

FIG. 46, illustrations (a)-(e), show various geometries of an opticalidentification element that may have holes therein, in accordance withthe present invention.

FIG. 47, illustrations (a)-(c), show various geometries of an opticalidentification element that may have teeth thereon, in accordance withthe present invention.

FIG. 48, illustrations (a)-(c), show various geometries of an opticalidentification element, in accordance with the present invention.

FIG. 49 is a side view an optical identification element having areflective coating thereon, in accordance with the present invention.

FIG. 50, illustrations (a)-(b), are side views of an opticalidentification element polarized along an electric or magnetic field, inaccordance with the present invention.

FIGS. 51 and 52 are diagrams of bead reads from flat retro-reflectortrays, in accordance with the present invention.

FIGS. 53 and 54 are diagrams of beads read thru V-grooves, in accordancewith the present invention.

FIGS. 55-83 are various alternative embodiments of the presentinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

FIG. 1 shows, by way of example, steps of a microbead assay processwhich uses the microbead technology of the present invention. The stepsof the assay process shown in FIG. 1 include a first step in which themicrobeads are used in a solution; a second step in which the microbeadsare aligned in a desired manner; a third step in which the code andflorescence in and/or on the microbeads are read-out; and a fourth stepin which the information related to the code and florescence isprocessed in relation to data management and bioinformatics. The presentinvention primarily relates to step 2 wherein the microbeads areuniquely aligned so the longitudinal axis of the microbeads ispositioned in a fixed orientation relative to the code and florescencereading device, as well as relating to a lesser extent to step 3. It isimportant to note that the scope of the present invention is notintended to be limited to any particular type or kind of assay processor other process in which the microbead technology is used. The scope ofthe invention is intended to include embodiments in which the microbeadtechnology of the present invention is used in many different processes.

Other processes/applications where the present invention may be usedinclude use of the beads in taggant applications, where the encodedbeads are used to identify, track, and/or authenticate, items such as isdiscussed in aforementioned copending U.S. patent application Ser. No.10/661,082, filed Sep. 12, 2003, entitled “Method and Apparatus forLabeling Using Diffraction Grating-Based Encoded Optical IdentificationElements”, (CyVera Docket No. CV-0040).

FIG. 2: The Microbead Element 8

FIG. 2 shows a diffraction grating-based optical identification element8 (or encoded element or coded element) that comprises a known opticalsubstrate 10, having an optical diffraction grating 12 disposed (orwritten, impressed, embedded, imprinted, etched, grown, deposited orotherwise formed) in the volume of or on a surface of the substrate 10along the length or longitudinal axis L of the element 8, which isotherwise known hereinafter as the microbead. The grating 12 is aperiodic or aperiodic variation in the effective refractive index and/oreffective optical absorption of at least a portion of the substrate 10.

The optical identification element 8 described herein is same as thatdescribed in Copending U.S. patent application Ser. No. 10/661,234,filed Sep. 12, 2003, entitled “Diffraction Grating-Based OpticalIdentification Element”, which is incorporated herein by reference inits entirety.

In particular, the substrate 10 has an inner region 20 where the grating12 is located. The inner region 20 may be photosensitive to allow thewriting or impressing of the grating 12. The substrate 10 has an outerregion 18, which does not have the grating 12 therein.

The grating 12 is a combination of one or more individual spatialperiodic sinusoidal variations (or components) in the refractive indexthat are collocated at substantially the same location on the substrate10 along the length of the grating region 20, each having a spatialperiod (or pitch) Λ. The resultant combination of these individualpitches is the grating 12, comprising spatial periods (Λ1-Λn) eachrepresenting a bit in the code. Thus, the grating 12 represents a uniqueoptically readable code, made up of bits, where a bit corresponds to aunique pitch Λ within the grating 12. Accordingly, for a digital binary(0-1) code, the code is determined by which spatial periods (Λ1-Λn)exist (or do not exist) in a given composite grating 12. The code orbits may also be determined by additional parameters (or additionaldegrees of multiplexing), and other numerical bases for the code may beused, as discussed herein and/or in the aforementioned patentapplication.

The grating 12 may also be referred to herein as a composite orcollocated grating. Also, the grating 12 may be referred to as a“hologram”, as the grating 12 transforms, translates, or filters aninput optical signal to a predetermined desired optical output patternor signal.

The substrate 10 has an outer diameter D1 and comprises silica glass(SiO₂) having the appropriate chemical composition to allow the grating12 to be disposed therein or thereon. Other materials for the opticalsubstrate 10 may be used if desired. For example, the substrate 10 maybe made of any glass, e.g., silica, phosphate glass, borosilicate glass,or other glasses, or made of glass and plastic, or solely plastic. Forhigh temperature or harsh chemical applications, the optical substrate10 made of a glass material is desirable. If a flexible substrate isneeded, plastic, rubber or polymer-based substrate may be used. Theoptical substrate 10 may be any material capable of having the grating12 disposed in the grating region 20 and that allows light to passthrough it to allow the code to be optically read.

The optical substrate 10 with the grating 12 has a length L and an outerdiameter D1, and the inner region 20 diameter D. The length L can rangefrom very small “microbeads” (or microelements, micro-particles, orencoded particles), about 1-1000 microns or smaller, to larger “macrobeads” or “macroelements” for larger applications (about 1.0-1000 mm orgreater). In addition, the outer dimension D1 can range from small (lessthan 1000 microns) to large (1.0-1000 mm and greater). Other dimensionsand lengths for the substrate 10 and the grating 12 may be used.

The grating 12 may have a length Lg of about the length L of thesubstrate 10. Alternatively, the length Lg of the grating 12 may beshorter than the total length L of the substrate 10.

The outer region 18 is made of pure silica (SiO₂) and has a refractiveindex n2 of about 1.458 (at a wavelength of about 1553 nm), and theinner grating region 20 of the substrate 10 has dopants, such asgermanium and/or boron, to provide a refractive index n1 of about 1.453,which is less than that of outer region 18 by about 0.005. Other indicesof refraction n1,n2 for the grating region 20 and the outer region 18,respectively, may be used, if desired, provided the grating 12 can beimpressed in the desired grating region 20. For example, the gratingregion 20 may have an index of refraction that is larger than that ofthe outer region 18 or grating region 20 may have the same index ofrefraction as the outer region 18 if desired.

FIG. 3: The Code Reader or Detector 29

FIG. 3 shows a configuration for reading or detecting the code in themicrobead 8 using a code reader or other detector device 29, which isused in step 3 of the process shown in FIG. 1. In operation, an incidentlight 24 of a wavelength λ, e.g., 532 nm from a known frequency doubledNd:YAG laser or 632 nm from a known Helium-Neon laser, is incident onthe grating 12 in the substrate 10. Any other input wavelength λ can beused if desired provided λ is within the optical transmission range ofthe substrate (discussed more herein and/or in the aforementioned patentapplication). A portion of the input light 24 passes straight throughthe grating 12, as indicated by a line 25. The remainder of the inputlight 24 is reflected by the grating 12, as indicated by a line 27 andprovided to a detector 29. The output light 27 may be a plurality ofbeams, each having the same wavelength λ as the input wavelength λ andeach having a different output angle indicative of the pitches (Λ1-Λn)existing in the grating 12. Alternatively, the input light 24 may be aplurality of wavelengths and the output light 27 may have a plurality ofwavelengths indicative of the pitches (Λ1-Λn) existing in the grating12. Alternatively, the output light may be a combination of wavelengthsand output angles. The above techniques are discussed in more detailherein and/or in the aforementioned patent application.

The code reader or detector 29 has the necessary optics, electronics,software and/or firmware to perform the functions described herein. Inparticular, the detector reads the optical signal 27 diffracted orreflected from the grating 12 and determines the code based on thepitches present or the optical pattern, as discussed more herein or inthe aforementioned patent application. An output signal indicative ofthe code is provided on a line 31.

The dimensions, geometries, materials, and material properties of thesubstrate 10 are selected such that the desired optical and materialproperties are met for a given application. The resolution and range forthe optical codes are scalable by controlling these parameters asdiscussed herein and/or in the aforementioned patent application. Also,the beads 8 may be made of any of the materials, geometries, andcoatings described in copending U.S. patent application Ser. No. (DocketNo. CV-0038A).

We have used the present invention with cylindrical beads having size ofabout 65 micron diameter and 400 microns long and about 28 micronsdiameter and about 250 microns long. However, other bead sizes may beused.

FIG. 4: The Grooved Tray or Plate

FIG. 4 shows one embodiment of a positioning device 200 for aligning themicrobeads 8 so the longitudinal axis of the microbeads is in a fixedorientation relative to the code reading or other detection device. Thepositioning device 200 is shown in the form of a tray or plate 200having v-grooves 205 for align the microbeads 8 and is used in step 2 ofthe process shown in FIG. 1.

As shown, the microbead elements 8 are placed in the tray 200 withv-grooves 205 to allow the elements 8 to be aligned in a predetermineddirection for illumination and reading/detection as discussed herein.Alternatively, the grooves 205 may have holes 210 that provide suctionto keep the elements 8 in position.

Forming the Grooves in the Groove Plate

The grooves in the groove plate may be made in many different ways,including being formed by SU8 photoresistant material, mechanicallymachining; deep reactive ion etching; or injection molding. Oneadvantage of the injection molding approach is that the plate can bemanufactured in volume at relatively low cost, and disposed of after theinformation about the beads is gathered in the assay process. The grooveplate may be made of glass, including fused silica, low fluorescenceglass, boro silicate glass, or other transparent glasses or plastic.Silicon is used because it is reflective so a reflective coating istypically not needed. Alternative, a mirror coating can be applied tothe plate material to achieve the desired reflectivity.

FIG. 5: Flat Grooves

The scope of the invention is not intended to be limited to anyparticular groove shape. For example, FIG. 5 shows a diagram a plate 300having flat grooves 302 instead of V-grooves as shown in FIG. 3. Somecharacteristics of the groove according to the present invention are asfollows:

The groove width (w) should be at least as wide as the diameter of thebead (D) but not larger than D+15 μm.

The thickness of the depth of the groove (T) should be at least 0.5times the diameter of the bead so that it sufficiently traps a bead onceit falls into the groove even when it is subjected to mechanicalagitation. The depth should not exceed 1.5 times the diameter of thebead so as to prevent more than one bead from falling into the samegroove location.

Groove plates have been made using a thick photoresist called SU8 and isavailable from Microchem. The resist is both chemically inert andmechanically robust once fully cured. The groove walls are formed by theresist material, which is deposited onto a glass or substrate.Advantages of this process include the ability to tailor the depth ofgroove by controlling the thickness of the resist material, andvirtually every other geometric attribute through the design of thephoto mask. Because it is photolithographic process, essentially anyshape profile can be made. For example grooves can be made in simplerows, concentric circles, or spirals. Other features such as discretewells, spots and cross hatches can be made as fiducial marks fortracking and positional registration purposes.

The scope of the invention is also intended to include the grooveshaving a flat bottom as shown in FIG. 5 with outwardly tapered walls.

FIG. 6: The Holey Plate 674

FIG. 6 shows an alternative embodiment, wherein alignment may beachieved by using a plate 674 having holes 676 slightly larger than theelements 8 if the light 24 (FIGS. 2 and 4) is incident along the gratingaxis 207. The incident light indicated as 670 is reflected off thegrating and exits through the end as a light 672 and the remaining lightpasses through the grating and the plate 674 as a line 678.Alternatively, if a blazed grating is used, incident light 670 may bereflected out the side of the plate (or any other desired angle), asindicated by a line 680. Alternatively, input light may be incident fromthe side of the plate 674 and reflected out the top of the plate 674 asindicated by a line 684. The light 670 may be a plurality of separatelight beams or a single light beam 686 that illuminates the entire tray674 if desired.

FIG. 7: V-Groove Plate 200 with End Illumination

FIG. 7 shows an alternative embodiment, wherein the v-groove platediscussed hereinbefore with FIG. 4 may be used for the endillumination/readout condition. In this case, the grating 12 may have ablaze angle such that light incident along the axial grating axis willbe reflected upward, downward, or at a predetermined angle for codedetection. Similarly, the input light may be incident on the grating ina downward, upward, or at a predetermined angle and the grating 12 mayreflect light along the axial grating axis for code detection.

FIG. 8: Microbead Mapper Readings

FIG. 8 shows microbeads 8 arranged on a plate 200 having grooves 205. Asshown, the microbeads 8 have different codes (e.g. “41101”, “20502”,“41125”) using 16-bit, binary symbology), which may be read or detectedusing the reader or detector configuration described in relation to FIG.3. The codes in the beads are used to provide a cross reference todetermine which probe is attached to which bead, thus allowing theresearcher to correlate the chemical content on each bead with themeasured fluorescence signal in Step 3 of the process shown in FIG. 1.

FIG. 8 a shows a code reader and detector for obtaining information fromthe microbead 8 in FIG. 8. The codes in the microbeads 8 are detectedwhen illuminated by incident light 24 which produces a diffracted oroutput light signal 27 to a reader 820, which includes the optics andelectronics necessary to read the codes in each bead 8, as describedherein and/or in the aforementioned copending patent application. Thereader 820 provides a signal on a line 822 indicative of the code ineach of the bead 8. The incident light 24 may be directed transverselyfrom the side of the grooved plate 200 (or from an end or any otherangle) with a narrow band (single wavelength) and/or multiple wavelengthsource, in which case the code is represented by a spatial distributionof light and/or a wavelength spectrum, respectively, as describedhereinafter and in the aforementioned copending patent application.Other illumination, readout techniques, types of gratings, geometries,materials, etc. may be used for the microbeads 8, as discussedhereinafter and in the aforementioned patent application.

For assays that use fluorescent molecule markers to label or tagchemicals, an optical excitation signal 800 is incident on themicrobeads 8 on the grooved plate 200 and a fluorescent optical outputsignal 802 emanates from the beads 8 that have the fluorescent moleculeattached. The fluorescent optical output signal 802 passes through alens 804, which provides focused light 802 to a known opticalfluorescence detector 808. Instead of or in addition to the lens 802,other imaging optics may be used to provide the desired characteristicsof the optical image/signal onto the fluorescence detector 808. Thedetector 808 provides an output signal on a line 810 indicative of theamount of fluorescence on a given bead 8, which can then be interpretedto determine what type of chemical is attached to the bead 10.

Consistent with that discussed herein, the grooved plate 200 may be madeof glass or plastic or any material that is transparent to the codereading incident beam 24 and code reading output light beams 27 as wellas the fluorescent excitation beam 800 and the output fluorescentoptical signal 802, and is properly suited for the desired applicationor experiment, e.g., temperature range, harsh chemicals, or otherapplication specific requirements.

The code signal 822 from the bead code reader 820 and the fluorescentsignal 810 from the fluorescence detector are provided to a knowncomputer 812. The computer 812 reads the code associated with each beadand determines the chemical probe that was attached thereto from apredetermined table that correlates a predetermined relationship betweenthe bead code and the attached probed. In addition, the computer 812 andreads the fluorescence associated with each bead and determines thesample or analyte that is attached to the bead from a predeterminedtable that correlates a predetermined relationship between thefluorescence tag and the analyte attached thereto. The computer 812 thendetermines information about the analyte and/or the probe as well asabout the bonding of the analyte to the probe, and provides suchinformation on a display, printout, storage medium or other interface toan operator, scientist or database for review and/or analysis,consistent with shown in step 4 of FIG. 1. The sources 801, 803 the codereader 820, the fluorescence optics 804 and detector 808 and thecomputer 812 may all be part of an assay stick reader 824.

Alternatively, instead of having the code excitation source 801 and thefluorescence excitation source 803, the reader 24 may have only onesource beam which provides both the reflected optical signal 27 fordetermining the code and the fluorescence signal 802 for reading thetagged analyte attached to the beads 8. In that case the input opticalsignal is a common wavelength that performs both functionssimultaneously, or sequentially, if desired.

The microbeads 8 may be coated with the desired probe compound,chemical, or molecule prior to being placed in the grooved plate 200.Alternatively, the beads 8 may be coated with the probe after beingplaced in the grooved plate 200. As discussed hereinbefore, the probematerial may be an Oligo, cDNA, polymer, or any other desired probecompound, chemical, cell, or molecule for performing an assay.

The scope of the invention is not intended to be limited to using ordetecting fluorescent molecule markers during the assay process. Forexample, embodiments of the invention are envisioned using and detectionother types of molecular markers in other types of processes.

Modes of Microbead Alignment

There are at least two possible modes or approaches of use for thegroove plate.

FIG. 9: Open Format Approach

FIG. 9 shows the first, or open plate format, meaning there is no top tocover the microbeads 8 and the v-grooves 205. In this mode, themicrobeads 8 are dispensed onto the plate 200 using, for example, apipette tip or syringe tip, although the scope of the invention is notintended to be limited to the manner of depositing the microbeads on theplate. The microbeads 8 may be then agitated by a sonic transducer (notshown), or manipulated with a mechanical wiper (not shown) or some formof spray nozzle (not shown) to encourage all the microbeads 8 to line upin the grooves 205. It has been observed that substantially all themicrobeads naturally line up in the grooves 205 without the need forencouragement. However, there are always some microbeads, such asmicrobead 8 a, 8 b, that do not fall naturally into the grooves, andthese must either be removed from the plate 200 or forced to fall into agroove 205. The open format approach has the advantages that groovesplate consists just of the plate and no other complicated features suchas walls and a top, and possibly other chambers or channels to allowfluid flow and bubble removal. It also has the advantage that it caneasily be made with a standard microscope slide, which is designed tofit conventional micro array readers or microscopes. However, the openformat approach would most likely require the microbeads to be dried outprior to reading, to prevent non-uniform and unpredictable opticalaberrations caused by the uneven evaporation of the buffer solution.

FIGS. 10-17: The Closed Format Approach

FIGS. 10-17 show the second mode which is called a closed format, thatconsists of not only of a groove plate but also a top and at least threewalls to hold the solution and the microbeads in a cuvette-like device(or cell or chamber) generally indicated as 500 shown, for example, inFIG. 10.

In summary, the closed format approach provides a method for effectivelydistributing and aligning microbeads during the readout process, asdescribed below:

The basic process for handling microbeads with a curvette for readoutconsists of the following steps:

(1) FIG. 10 shows a starting point for handling microbeads for areadout. The microbeads start in a test tube. Typical test-tube volumesare 1.5 ml. The microbeads will generally be in a liquid (usually waterwith a small amount of other buffer chemicals to adjust pH and possiblya small amount [˜0.01%] of detergent.) As shown, a bead tube 502contains the microbeads in a solution, which forms part of step 1 of theprocess shown in FIG. 1.

(2) FIG. 11 shows the bead tube 502 is coupled to a flange 504 of thecuvette 500 is inverted and the beads flow onto the groove plate. Thecuvette consists of two round flanges that accept test-tubes, atransparent window, and an opposing groove plate. FIG. 14 shows adrawing of a prototype cuvette. The groove plate outer dimensions can beany size, but typical microscope slide dimensions are convenient(1″×3″). The grooves are mechanically or laser cut lengthwise, and havedimensions that are chosen for the exact size of cylindrical microbead.For instance, for a 125 μm diameter bead, grooves of approximately 150μm wide by 150 μm deep are used. One tube carries the microbeads and asmall amount of carrier fluid. The second tube may be larger and holdmore fluid. The purpose of the second tube is to guarantee a certainfluid level in the next step.

(3) After the cuvette is inverted and the microbeads flow out onto thegroove plate side of the cuvette, the microbeads naturally align in thegrooves via a small amount of rocking or agitation, which forms part ofstep 2 of the process shown in FIG. 1.

(4) FIG. 12 shows the readout step, in which, after the beads are all(or nearly all) aligned in the groove plate, the entire plate is moved(or the readout laser beam is scanned) in order to read the codes ofeach beam, which forms part of step 3 of the process shown in FIG. 1. Ineffect, once the microbeads are in the grooves, the entire cuvette ismoved back and forth across a readout beam. The readout beam istransmitted through the cuvette and contains the code bits encoded onthe scattering angles.

(5) FIG. 13 shows a final step, in which the cuvette is inverted to itsoriginal position and the beads flow back into the original tube 502,which forms part of step 3 of the process shown in FIG. 1. In otherwords, after the readout process, the cuvette is re-inverted and themicrobeads flow back into the original test tube.

FIG. 14 shows an example of a cuvette generally indicated as 700 that ismounted on a kinematic base plate 710. As shown, the cuvette 700 has atube 702 for holding the solution with the beads and a top window 704that is a 1 mm thick glass plate having dimensions of about 1″ by 3″.The cuvette also has a bottom plate that is a transparent groove plate.The location pins 712 and lever arm 714 hold the cuvette 700 in place onthe kinematic plate 710.

One of the key advantages of using the cuvette device is that thepotential to nearly index match the glass microbeads with a buffersolution thereby reducing the divergence of the laser beam caused by thelensing effect of the microbeads, and minimizing scatter form the grooveplate itself.

Another advantage involves the potential to prevent microbeads from everstacking up on top of each other, by limiting the space between thebottom and the top plate to be less than twice the diameter of themicrobeads.

Another advantage is that the cover keeps the fluid from evaporating.

FIGS. 15-16

FIGS. 15-16 show alternative embodiments of the cuvette shown in FIGS.10-14. As shown, the microbeads are injected into the cuvette by placingthem near the edge of the opening and allowing the surface tension, oran induced fluid flow, to pull the microbeads into the cuvette, where,because of the limited height between the floor and the ceiling of thecuvette, they are confined to move around in a plane, albeit with allthe rotational degrees of freedom unconstrained. Once in the cuvette themicrobeads are quickly and sufficiently constrained by the grooves asthe microbeads fall into them. As in the case of the open format thereis still the finite probability that some number of microbeads will notfall into the grooves and must be coaxed in by some form of agitation(ultrasonic, shaking, rocking, etc.).

FIG. 17: Two Region Approach

FIG. 17 shows an alternative embodiment of the closed approach, whichinvolves sectioning the closed region into two regions, one where themicrobeads are free to move about in a plane, either in a groove or not,and a second region where the microbeads are trapped in a groove and canonly move along the axes of a groove. Trapping the microbeads in agroove is accomplished by further reducing the height of the chamber tothe extent that the microbeads can no longer hop out of a groove. Inthis embodiment, the free region is used to pre-align the microbeadsinto a groove, facilitating the introduction of microbeads into thetrapped section. By tilting this type of cuvette up gravity can be usedto pull the microbeads along a groove from the free region to thetrapped region. Once in the trapped region the microbeads move to theend of the groove where they stop. Subsequent microbeads will begin tostack up until the groove is completely full of microbeads, which arestacked head to tail. This has the advantage of packing a large numberof microbeads into a small area and prevents the microbeads from everjumping out of the grooves. This approach could also be used to alignthe microbeads prior to injection into some form of flow cytometer, or adispensing apparatus.

FIGS. 18-23: The Cytometer

FIGS. 18-23 show method and apparatus related to using a cytometer.

FIG. 18(a) shows steps for a method related to a conventional (singlepass) flow cytometer reader and FIG. 18(b) shows a method related to adisk cytometer reader (multipass).

In FIG. 18(a), the method generally indicated as 900 has a step forproviding beads and a solution similar to step 1 in FIG. 1; and a stepfor reading information from the beads similar to steps 2 and 3 in FIG.1.

In FIG. 18(b), the method generally indicated as 1000 has a step forproviding beads and solution similar to step 1 in FIG. 1; and a step forspinning and reading information from the beads similar to steps 2 and 3in FIG. 1.

In the methods shown in FIGS. 18(a) and (b), a rotating disk (see FIGS.19(a), (b) and (c) and 20) is used for aligning the microbeadsconsistent with step 2 of the process shown in FIG. 1.

FIG. 19(a) shows an embodiment of a cytometer bead reader having arotating disk generally indicated as 1250, having a disk platform 1252with circumferential, concentric, grooves 1254 for aligning microbeads8. As shown, the rotating disk 1250 has various sectors for processingthe microbeads, including a bead loading zone 1256, a bead removal zone1258 and a readout zone 1260, as well as a barrier 1259 for preventingthe microbeads from flying off the plate. As shown, a window 1262 forreading the beads is in contact with the fluid containing the beads.

FIG. 19(b) shows an alternative embodiment of a rotating disk generallyindicated as 1200, having a disk platform 1202 with planar groove plates1204 a, b, c, d, e, f that are shown with grooves oriented in any one ormore different ways. One or more of the planar groove plates 1204 a, b,c, d, e, f may have an optional channel for fluid run-off, as shown, anda barrier (FIG. 19(a)) for preventing the microbeads from flying off theplate. All other attributes may be the same as described in FIG. 19(a).A window 1263 may be used for loading and/or reading the beads on thegroove plates 1204 a, b, c, d, e, f.

FIG. 19(c) shows an alternative embodiment of a rotating disk generallyindicated as 1280, having a disk platform 1282 with radial grooves 1284a, 1284 b. The disk platform 1282 has a bead loading zone 1286 in thecenter of the disk. One advantage of this embodiment is that the openingof the bead loading zone 1286 will also serve to allow the release ofair bubbles that will naturally collect in the center of the disk duethe reduced density of the fluid, which results from the centrifugalforce pushing the fluid radially outwardly. The rotating disk 1280 hastight bead packing due to the centrifugal forces due to the spinningaction of the disk. The rotating disk 1280 has a wedge shape spacer 1288that keeps the channel at a constant gap width and a wall 1290.

FIG. 20(a) shows an alternative embodiment of a rotating disk generallyindicated as 1300 having narrow radial channels 1302 for spin drying soliquid is forced out of the circumferential grooves through the radialchannels. The plate 1300 may have a mechanical catcher 1320 coupledthereto for moving radially outwardly in direction 1320 a if desired,for recirculating loose beads.

FIG. 20(b) show an alternative embodiment of a disk cytometer 1400having a mechanical iris 1402 for providing a variable aperture for beadaccess to grooves in accordance with the invention.

FIG. 21 shows a rotating groove plate having 450 by 65 microns beadsarranged in the rotating SU8 circumferential channels.

For any of the circular groove plates shown herein, the disk may rotateas discussed above and/or the reader excitation laser(s)/detector(s) mayrotate to read the code and/or the fluorescence on the beads 8.

Continuous Mode—Process Steps

The following are the processing steps for a continuous mode ofoperation:

1. Dispense batch of microbeads onto plate.

2. Spin slowly while agitating the plate theta x and y to get microbeadsinto grooves. The agitation can be performed using rocking, ultrasound,airflow, etc.

3. Once sufficient number of microbeads are in grooves, spin up plate toremove excess microbeads (microbeads that did not go into a groove).

4. Spin disk to read code and fluorescence.

5. To remove microbeads, purge with high velocity aqueous solution(enough to knock microbeads out of groove) and vacuum up, or spinmicrobeads off plate while they are not in a groove.

6. Inspect disk (probably with code camera) to verify that allmicrobeads have been removed.

7. Inject next batch of microbeads.

FIGS. 22-23: The Alignment Tube 502

In FIG. 22, instead of a flat grooved plate 200 (FIG. 3), the microbeadsmay be aligned in a tube 502 that has a diameter that is only slightlylarger than the substrate 10, e.g., about 1-50 microns, and that issubstantially transparent to the incident light 24. In that case, theincident light 24 may pass through the tube 502 as indicated by thelight 500 or be reflected back due to a reflective coating on the tube502 or the substrate as shown by return light 504. Other techniques canbe used for alignment if desired.

FIG. 23 shows the tube 502 has an opening flange 512 for receiving themicrobeads. FIG. 23 also shows an excitation laser 550, a diode laser552 and a CCD camera 554 for gathering information from the bead 8consistent with that discussed above. If desired, the beads 8 may bealigned and flowed through the tube 502 (similar to that discussed withFIG. 18(a) flow cytometer). In that case, fluid (liquid and/or gas) mayflow through the tube 508 to move the beads 8 along the tube 502, usinga flow cytometer approach.

FIGS. 24-44: Reading the Microbead Code and Alternative Embodiments

FIGS. 24-44 provide a method and apparatus for reading the code in themicrobeads 8, as well as a more detailed description of the microbeads 8and certain alternative embodiments therefore. The scope of theinvention is not intended to be limited in any way to the manner inwhich the code is read, or the method of doing the same.

Referring to FIG. 24, The reflected light 27, comprises a plurality ofbeams 26-36 that pass through a lens 37, which provides focused lightbeams 46-56, respectively, which are imaged onto a CCD camera 60. Thelens 37 and the camera 60, and any other necessary electronics or opticsfor performing the functions described herein, make up the reader 29.Instead of or in addition to the lens 37, other imaging optics may beused to provide the desired characteristics of the optical image/signalonto the camera 60 (e.g., spots, lines, circles, ovals, etc.), dependingon the shape of the substrate 10 and input optical signals. Also,instead of a CCD camera other devices may be used to read/capture theoutput light.

Referring to FIG. 25, the image on the CCD camera 60 is a series ofilluminated stripes indicating ones and zeros of a digital pattern orcode of the grating 12 in the element 8. Referring to FIG. 26, lines 68on a graph 70 are indicative of a digitized version of the image of FIG.25 as indicated in spatial periods (Λ1-Λn).

Each of the individual spatial periods (Λ1-Λn) in the grating 12 isslightly different, thus producing an array of N unique diffractionconditions (or diffraction angles) discussed more hereinafter. When theelement 8 is illuminated from the side, in the region of the grating 12,at an appropriate input angle, e.g., about 30 degrees, with a singleinput wavelength λ (monochromatic) source, the diffracted (or reflected)beams 26-36 are generated. Other input angles θi may be used if desired,depending on various design parameters as discussed herein and/or in theaforementioned patent application, and provided that a known diffractionequation (Eq. 1 below) is satisfied:sin(θ_(i))+sin(θ_(o))=mλ/nΛ  Eq. 1where Eq. 1 is diffraction (or reflection or scatter) relationshipbetween input wavelength λ, input incident angle θi, output incidentangle θo, and the spatial period Λ of the grating 12. Further, m is the“order” of the reflection being observed, and n is the refractive indexof the substrate 10. The value of m=1 or first order reflection isacceptable for illustrative purposes. Eq. 1 applies to light incident onouter surfaces of the substrate 10 which are parallel to thelongitudinal axis of the grating (or the k_(B) vector). Because theangles θi,θo are defined outside the substrate 10 and because theeffective refractive index of the substrate 10 is substantially a commonvalue, the value of n in Eq. 1 cancels out of this equation.

Thus, for a given input wavelength λ, grating spacing Λ, and incidentangle of the input light θi, the angle θo of the reflected output lightmay be determined. Solving Eq. 1 for θo and plugging in m=1, gives:θo=sin⁻¹(λ/Λ−sin(θi))  Eq. 2For example, for an input wavelength λ=532 nm, a grating spacing Λ=0.532microns (or 532 nm), and an input angle of incidence θi=30 degrees, theoutput angle of reflection will be θo=30 degrees. Alternatively, for aninput wavelength λ=632 nm, a grating spacing Λ=0.532 microns (or 532nm), and an input angle θi of 30 degrees, the output angle of reflectionθo will be at 43.47 degrees, or for an input angle θi=37 degrees, theoutput angle of reflection will be θo=37 degrees. Any input angle thatsatisfies the design requirements discussed herein and/or in theaforementioned patent application may be used.

In addition, to have sufficient optical output power and signal to noiseratio, the output light 27 should fall within an acceptable portion ofthe Bragg envelope (or normalized reflection efficiency envelope) curve200, as indicated by points 204,206, also defined as a Bragg envelopeangle θB, as also discussed herein and/or in the aforementioned patentapplication. The curve 200 may be defined as: $\begin{matrix}{{I\left( {{ki},{ko}} \right)} \approx {\lbrack{KD}\rbrack^{2}\sin\quad{c^{2}\left\lbrack \frac{\left( {{ki} - {ko}} \right)D}{2} \right\rbrack}}} & {{Eq}.\quad 3}\end{matrix}$where K=2πδn/λ, where, δn is the local refractive index modulationamplitude of the grating and λ is the input wavelength,sinc(x)=sin(x)/x, and the vectors k_(i)=2πcos(θ_(i))/λ and k_(o)=2πcos(θ_(o))/λ are the projections of the incident light and the output (orreflected) light, respectively, onto the line 203 normal to the axialdirection of the grating 12 (or the grating vector k_(B)), D is thethickness or depth of the grating 12 as measured along the line 203(normal to the axial direction of the grating 12). Other substrateshapes than a cylinder may be used and will exhibit a similar peakedcharacteristic of the Bragg envelope. We have found that a value for δnof about 10⁻⁴ in the grating region of the substrate is acceptable;however, other values may be used if desired.

Rewriting Eq. 3 gives the reflection efficiency profile of the Braggenvelope as: $\begin{matrix}{{{I\left( {{ki},{ko}} \right)} \approx {{\left\lbrack \frac{2{\pi \cdot \delta}\quad{n \cdot D}}{\lambda} \right\rbrack^{2}\left\lbrack \frac{{Sin}(x)}{x} \right\rbrack}^{2}\quad\text{where:}}}{x = {{\left( {{ki} - {ko}} \right){D/2}} = {\left( {\pi\quad{D/\lambda}} \right)*\left( {{\cos\quad\theta\quad i} - {\cos\quad\theta\quad o}} \right)}}}} & {{Eq}.\quad 4}\end{matrix}$

Thus, when the input angle θi is equal to the output (or reflected)angle θ_(o) (i.e., θi=θ_(o)), the reflection efficiency I (Eqs. 3 & 4)is maximized, which is at the center or peak of the Bragg envelope. Whenθi=θo, the input light angle is referred to as the Bragg angle as isknown. The efficiency decreases for other input and output angles (i.e.,θi # θ), as defined by Eqs. 3 & 4. Thus, for maximum reflectionefficiency and thus output light power, for a given grating pitch Λ andinput wavelength, the angle θi of the input light 24 should be set sothat the angle θo of the reflected output light equals the input angleθi.

Also, as the thickness or diameter D of the grating decreases, the widthof the sin(x)/x function (and thus the width of the Bragg envelope)increases and, the coefficient to or amplitude of the sinc² (or(sin(x)/x)² function (and thus the efficiency level across the Braggenvelope) also increases, and vice versa. Further, as the wavelength λincreases, the half-width of the Bragg envelope as well as theefficiency level across the Bragg envelope both decrease. Thus, there isa trade-off between the brightness of an individual bit and the numberof bits available under the Bragg envelope. Ideally, δn should be madeas large as possible to maximize the brightness, which allows D to bemade smaller.

From Eq. 3 and 4, the half-angle of the Bragg envelope θ_(B) is definedas: $\begin{matrix}{\theta_{B} = \frac{\eta\quad\lambda}{\pi\quad D\quad{\sin\left( \theta_{i} \right)}}} & {{Eq}.\quad 5}\end{matrix}$where η is a reflection efficiency factor which is the value for x inthe sinc²(x) function where the value of sinc²(x) has decreased to apredetermined value from the maximum amplitude as indicated by points204,206 on the curve 200.

We have found that the reflection efficiency is acceptable when η≦1.39.This value for η corresponds to when the amplitude of the reflected beam(i.e., from the sinc²(x) function of Eqs. 3 & 4) has decayed to about50% of its peak value. In particular, when x=1.39=η, sinc²(x)=0.5.However, other values for efficiency thresholds or factor in the Braggenvelope may be used if desired.

The beams 26-36 are imaged onto the CCD camera 60 to produce the patternof light and dark regions 120-132 representing a digital (or binary)code, where light=1 and dark=0 (or vice versa). The digital code may begenerated by selectively creating individual index variations (orindividual gratings) with the desired spatial periods Λ1-Λn. Otherillumination, readout techniques, types of gratings, geometries,materials, etc. may be used as discussed in the aforementioned patentapplication.

Referring to FIG. 26, illustrations (a)-(c), for the grating 12 in acylindrical substrate 10 having a sample spectral 17 bit code (i.e., 17different pitches Λ1-Λ17), the corresponding image on the CCD (ChargeCoupled Device) camera 60 is shown for a digital pattern of 7 bitsturned on (10110010001001001); 9 bits turned on of (1000101010100111);all 17 bits turned on of (11111111111111111).

For the images in FIG. 26, the length of the substrate 10 was 450microns, the outer diameter D1 was 65 microns, the inner diameter D was14 microns, δn for the grating 12 was about 10⁻⁴, n1 in portion 20 wasabout 1.458 (at a wavelength of about 1550 nm), n2 in portion 18 wasabout 1.453, the average pitch spacing Λ for the grating 12 was about0.542 microns, and the spacing between pitches ΔΛ was about 0.36% of theadjacent pitches Λ.

Referring to FIG. 27, illustration (a), the pitch Λ of an individualgrating is the axial spatial period of the sinusoidal variation in therefractive index n1 in the region 20 of the substrate 10 along the axiallength of the grating 12 as indicated by a curve 90 on a graph 91.Referring to FIG. 27, illustration (b), a sample composite grating 12comprises three individual gratings that are co-located on the substrate10, each individual grating having slightly different pitches, Λ1, Λ2,Λ3, respectively, and the difference (or spacing) ΔΛ between each pitchΛ being about 3.0% of the period of an adjacent pitch Λ as indicated bya series of curves 92 on a graph 94. Referring to FIG. 27, illustration(c), three individual gratings, each having slightly different pitches,Λ1, Λ2, Λ3, respectively, are shown, the difference AA between eachpitch Λ being about 0.3% of the pitch Λ of the adjacent pitch as shownby a series of curves 95 on a graph 97. The individual gratings in FIG.27, illustrations (b) and (c) are shown to all start at 0 forillustration purposes; however, it should be understood that, theseparate gratings need not all start in phase with each other. Referringto FIG. 27, illustration (d), the overlapping of the individualsinusoidal refractive index variation pitches Λ1-Λn in the gratingregion 20 of the substrate 10, produces a combined resultant refractiveindex variation in the composite grating 12 shown as a curve 96 on agraph 98 representing the combination of the three pitches shown in FIG.27, illustration (b). Accordingly, the resultant refractive indexvariation in the grating region 20 of the substrate 10 may not besinusoidal and is a combination of the individual pitches Λ (or indexvariation).

The maximum number of resolvable bits N, which is equal to the number ofdifferent grating pitches Λ (and hence the number of codes), that can beaccurately read (or resolved) using side-illumination and side-readingof the grating 12 in the substrate 10, is determined by numerousfactors, including: the beam width w incident on the substrate (and thecorresponding substrate length L and grating length Lg), the thicknessor diameter D of the grating 12, the wavelength λ of incident light, thebeam divergence angle θ_(R), and the width of the Bragg envelope θ_(B)(discussed more in the aforementioned patent application), and may bedetermined by the equation: $\begin{matrix}{N \cong \frac{\eta\quad\beta\quad L}{2\quad D\quad{\sin\left( \theta_{i} \right)}}} & {{Eq}.\quad 6}\end{matrix}$

Referring to FIG. 28, instead of having the input light 24 at a singlewavelength λ (monochromatic) and reading the bits by the angle θo of theoutput light, the bits (or grating pitches Λ) may be read/detected byproviding a plurality of wavelengths and reading the wavelength spectrumof the reflected output light signal. In this case, there would be onebit per wavelength, and thus, the code is contained in the wavelengthinformation of the reflected output signal.

In this case, each bit (or Λ) is defined by whether its correspondingwavelength falls within the Bragg envelope, not by its angular positionwithin the Bragg envelope 200. As a result, it is not limited by thenumber of angles that can fit in the Bragg envelope 200 for a givencomposite grating 12, as in the embodiment discussed hereinbefore. Thus,using multiple wavelengths, the only limitation in the number of bits Nis the maximum number of grating pitches Λ that can be superimposed andoptically distinguished in wavelength space for the output beam.

Referring to FIGS. 28 and 29, illustration (a), the reflectionwavelength spectrum (λ1-λn) of the reflected output beam 310 willexhibit a series of reflection peaks 695, each appearing at the sameoutput Bragg angle θo. Each wavelength peak 695 (λ1-λn) corresponds toan associated spatial period (Λ1-Λn), which make up the grating 12.

One way to measure the bits in wavelength space is to have the inputlight angle θi equal to the output light angle θo, which is kept at aconstant value, and to provide an input wavelength λ that satisfies thediffraction condition (Eq. 1) for each grating pitch Λ. This willmaximize the optical power of the output signal for each pitch Λdetected in the grating 12.

Referring to 29, illustration (b), the transmission wavelength spectrumof the transmitted output beam 330 (which is transmitted straightthrough the grating 12) will exhibit a series of notches (or dark spots)696. Alternatively, instead of detecting the reflected output light 310,the transmitted light 330 may be detected at the detector/reader 308. Itshould be understood that the optical signal levels for the reflectionpeaks 695 and transmission notches 696 will depend on the “strength” ofthe grating 12, i.e., the magnitude of the index variation n in thegrating 12.

In FIG. 28, the bits may be detected by continuously scanning the inputwavelength. A known optical source 300 provides the input light signal24 of a coherent scanned wavelength input light shown as a graph 304.The source 300 provides a sync signal on a line 306 to a known reader308. The sync signal may be a timed pulse or a voltage ramped signal,which is indicative of the wavelength being provided as the input light24 to the substrate 10 at any given time. The reader 308 may be aphotodiode, CCD camera, or other optical detection device that detectswhen an optical signal is present and provides an output signal on aline 309 indicative of the code in the substrate 10 or of thewavelengths present in the output light, which is directly related tothe code, as discussed herein. The grating 12 reflects the input light24 and provides an output light signal 310 to the reader 308. Thewavelength of the input signal is set such that the reflected outputlight 310 will be substantially in the center 314 of the Bragg envelope200 for the individual grating pitch (or bit) being read.

Alternatively, the source 300 may provide a continuous broadbandwavelength input signal such as that shown as a graph 316. In that case,the reflected output beam 310 signal is provided to a narrow bandscanning filter 318 which scans across the desired range of wavelengthsand provides a filtered output optical signal 320 to the reader 308. Thefilter 318 provides a sync signal on a line 322 to the reader, which isindicative of which wavelengths are being provided on the output signal320 to the reader and may be similar to the sync signal discussedhereinbefore on the line 306 from the source 300. In this case, thesource 300 does not need to provide a sync signal because the inputoptical signal 24 is continuous. Alternatively, instead of having thescanning filter being located in the path of the output beam 310, thescanning filter may be located in the path of the input beam 24 asindicated by the dashed box 324, which provides the sync signal on aline 323.

Alternatively, instead of the scanning filters 318,324, the reader 308may be a known optical spectrometer (such as a known spectrum analyzer),capable of measuring the wavelength of the output light.

The desired values for the input wavelengths λ (or wavelength range) forthe input signal 24 from the source 300 may be determined from the Braggcondition of Eq. 1, for a given grating spacing Λ and equal angles forthe input light θi and the angle light θo. Solving Eq. 1 for λ andplugging in m=1, gives:λ=Λ[sin(θo)+sin(θi)]  Eq. 7

It is also possible to combine the angular-based code detection with thewavelength-based code detection, both discussed hereinbefore. In thiscase, each readout wavelength is associated with a predetermined numberof bits within the Bragg envelope. Bits (or grating pitches Λ) writtenfor different wavelengths do not show up unless the correct wavelengthis used.

Accordingly, the bits (or grating pitches Λ) can be read using onewavelength and many angles, many wavelengths and one angle, or manywavelengths and many angles.

Referring to FIG. 30, the grating 12 may have a thickness or depth Dwhich is comparable or smaller than the incident beam wavelength λ. Thisis known as a “thin” diffraction grating (or the full angle Braggenvelope is 180 degrees). In that case, the half-angle Bragg envelope θBis substantially 90 degrees; however, δn must be made large enough toprovide sufficient reflection efficiency, per Eqs. 3 and 4. Inparticular, for a “thin” grating, D*δn≈λ/2, which corresponds to a πphase shift between adjacent minimum and maximum refractive index valuesof the grating 12.

It should be understood that there is still a trade-off discussedhereinbefore with beam divergence angle θ_(R) and the incident beamwidth (or length L of the substrate), but the accessible angular spaceis theoretically now 90 degrees. Also, for maximum efficiency, the phaseshift between adjacent minimum and maximum refractive index values ofthe grating 12 should approach a π phase shift; however, other phaseshifts may be used.

In this case, rather than having the input light 24 coming in at theconventional Bragg input angle θi, as discussed hereinbefore andindicated by a dashed line 701, the grating 12 is illuminated with theinput light 24 oriented on a line 705 orthogonal to the longitudinalgrating vector 705. The input beam 24 will split into two (or more)beams of equal amplitude, where the exit angle θ_(o) can be determinedfrom Eq. 1 with the input angle θ_(i)=0 (normal to the longitudinal axisof the grating 12).

In particular, from Eq. 1, for a given grating pitch Λ1, the +/−1^(st)order beams (m=++1 and m=−1), corresponds to output beams 700,702,respectively. For the +/−2^(nd) order beams (m=+2 and m=−2), correspondsto output beams 704,706, respectively. The 0^(th) order (undefracted)beam (m=0), corresponds to beam 708 and passes straight through thesubstrate. The output beams 700-708 project spectral spots or peaks710-718, respectively, along a common plane, shown from the side by aline 709, which is parallel to the upper surface of the substrate 10.

For example, for a grating pitch Λ=1.0 um, and an input wavelength λ=400nm, the exit angles θ_(o) are ˜+/−23.6 degrees (for m=+/−1), and +/−53.1degrees (from m=+/−2), from Eq. 1. It should be understood that forcertain wavelengths, certain orders (e.g., m=+/−2) may be reflected backtoward the input side or otherwise not detectable at the output side ofthe grating 12.

Alternatively, one can use only the +/−1^(st) order (m=+/−1) outputbeams for the code, in which case there would be only 2 peaks to detect,712, 714. Alternatively, one can also use any one or more pairs from anyorder output beam that is capable of being detected. Alternatively,instead of using a pair of output peaks for a given order, an individualpeak may be used.

Referring to FIG. 31, if two pitches Λ1,Λ2 exist in the grating 12, twosets of peaks will exist. In particular, for a second grating pitch Λ2,the +/−1^(st) order beams (m=+1 and m=−1), corresponds to output beams720,722, respectively. For the +/−2^(nd) order beams (m=+2 and m=−2),corresponds to output beams 724,726, respectively. The 0^(th) order(un-defracted) beam (m=0), corresponds to beam 718 and passes straightthrough the substrate. The output beams 720-726 corresponding to thesecond pitch Λ2 project spectral spots or peaks 730-736, respectively,which are at a different location than the point 710-716, but along thesame common plane, shown from the side by the line 709.

Thus, for a given pitch Λ (or bit) in a grating, a set of spectral peakswill appear at a specific location in space. Thus, each different pitchcorresponds to a different elevation or output angle which correspondsto a predetermined set of spectral peaks. Accordingly, the presence orabsence of a particular peak or set of spectral peaks defines the code.

In general, if the angle of the grating 12 is not properly aligned withrespect to the mechanical longitudinal axis of the substrate 10, thereadout angles may no longer be symmetric, leading to possibledifficulties in readout. With a thin grating, the angular sensitivity tothe alignment of the longitudinal axis of the substrate 10 to the inputangle θi of incident radiation is reduced or eliminated. In particular,the input light can be oriented along substantially any angle θi withrespect to the grating 12 without causing output signal degradation, duethe large Bragg angle envelope. Also, if the incident beam 24 is normalto the substrate 10, the grating 12 can be oriented at any rotational(or azimuthal) angle without causing output signal degradation. However,in each of these cases, changing the incident angle θi will affect theoutput angle θo of the reflected light in a predetermined predictableway, thereby allowing for accurate output code signal detection orcompensation.

Referring to FIG. 32, for a thin grating, in addition to multiplexing inthe elevation or output angle based on grating pitch Λ, the bits canalso be multiplexed in an azimuthal (or rotational) angle θa of thesubstrate. In particular, a plurality of gratings 750,752,754,756 eachhaving the same pitch Λ are disposed in a surface 701 of the substrate10 and located in the plane of the substrate surface 701. The inputlight 24 is incident on all the gratings 750,752,754,756 simultaneously.Each of the gratings provides output beams oriented based on the gratingorientation. For example, the grating 750 provides the output beams764,762, the grating 752 provides the output beams 766,768, the grating754 provides the output beams 770,772, and the grating 756 provides theoutput beams 774,776. Each of the output beams provides spectral peaksor spots (similar to that discussed hereinbefore), which are located ina plane 760 that is parallel to the substrate surface plane 701. In thiscase, a single grating pitch Λ can produce many bits depending on thenumber of gratings that can be placed at different azimuthal(rotational) angles on the surface of the substrate 10 and the number ofoutput beam spectral peaks that can be spatially and opticallyresolved/detected. Each bit may be viewed as the presence or absence ofa pair of peaks located at a predetermined location in space in theplane 760. Note that this example uses only the m=+/−1^(st) order foreach reflected output beam. Alternatively, the detection may also usethe m=+/−2^(nd) order. In that case, there would be two additionaloutput beams and peaks (not shown) for each grating (as discussedhereinbefore) that may lie in the same plane as the plane 760 and may beon a concentric circle outside the circle 760.

In addition, the azimuthal multiplexing can be combined with theelevation or output angle multiplexing discussed hereinbefore to providetwo levels of multiplexing. Accordingly, for a thin grating, the numberof bits can be multiplexed based on the number of grating pitches Λand/or geometrically by the orientation of the grating pitches.

Furthermore, if the input light angle θi is normal to the substrate 10,the edges of the substrate 10 no longer scatter light from the incidentangle into the “code angular space”, as discussed herein and/or in theaforementioned patent application.

Also, in the thin grating geometry, a continuous broadband wavelengthsource may be used as the optical source if desired.

Referring to FIG. 33, instead of or in addition to the pitches Λ in thegrating 12 being oriented normal to the longitudinal axis, the pitchesmay be created at a angle θg. In that case, when the input light 24 isincident normal to the surface 792, will produce a reflected output beam790 having an angle θo determined by Eq. 1 as adjusted for the blazeangle θg. This can provide another level of multiplexing bits in thecode.

Referring to FIG. 34, instead of using an optical binary (0-1) code, anadditional level of multiplexing may be provided by having the opticalcode use other numerical bases, if intensity levels of each bit are usedto indicate code information. This could be achieved by having acorresponding magnitude (or strength) of the refractive index change(δn) for each grating pitch Λ. Four intensity ranges are shown for eachbit number or pitch Λ, providing for a Base-4 code (where each bitcorresponds to 0,1,2, or 3). The lowest intensity level, correspondingto a 0, would exist when this pitch Λ is not present in the grating 12.The next intensity level 450 would occur when a first low level δn1exists in the grating that provides an output signal within theintensity range corresponding to a 1. The next intensity level 452 wouldoccur when a second higher level δn2 exists in the grating 12 thatprovides an output signal within the intensity range corresponding to a2. The next intensity level 452, would occur when a third higher levelδn3 exists in the grating 12 that provides an output signal within theintensity range corresponding to a 3.

Referring to FIG. 35, the input light 24 may be incident on thesubstrate 10 on an end face 600 of the substrate 10. In that case, theinput light 24 will be incident on the grating 12 having a moresignificant component of the light (as compared to side illuminationdiscussed hereinbefore) along the longitudinal grating axis 207 of thegrating (along the grating vector k_(B)), as shown by a line 602. Thelight 602 reflects off the grating 12 as indicated by a line 604 andexits the substrate as output light 608. Accordingly, it should beunderstood by one skilled in the art that the diffraction equationsdiscussed hereinbefore regarding output diffraction angle θo also applyin this case except that the reference axis would now be the gratingaxis 207. Thus, in this case, the input and output light angles θi,θo,would be measured from the grating axis 207 and length Lg of the grating12 would become the thickness or depth D of the grating 12. As a result,a grating 12 that is 400 microns long, would result in the Braggenvelope 200 being narrow. It should be understood that because thevalues of n1 and n2 are close to the same value, the slight anglechanges of the light between the regions 18,20 are not shown herein.

In the case where incident light 610 is incident along the samedirection as the grating vector (Kb) 207, i.e., θi=0 degrees, theincident light sees the whole length Lg of the grating 12 and thegrating provides a reflected output light angle θo=0 degrees, and theBragg envelope 612 becomes extremely narrow, as the narrowing effectdiscussed above reaches a limit. In that case, the relationship betweena given pitch Λ in the grating 12 and the wavelength of reflection λ isgoverned by a known “Bragg grating” relation:λ=2 n _(eff) Λ  Eq. 8where n_(eff) is the effective index of refraction of the substrate, λis the input (and output wavelength) and Λ is the pitch. This relation,as is known, may be derived from Eq. 1 where θi=θo=90 degrees.

In that case, the code information is readable only in the spectralwavelength of the reflected beam, similar to that discussed hereinbeforefor wavelength based code reading. Accordingly, the input signal in thiscase may be a scanned wavelength source or a broadband wavelengthsource. In addition, as discussed hereinbefore for wavelength based codereading, the code information may be obtained in reflection from thereflected beam 614 or in transmission by the transmitted beam 616 thatpasses through the grating 12.

It should be understood that for shapes of the substrate 10 or element 8other than a cylinder, the effect of various different shapes on thepropagation of input light through the element 8, substrate 10, and/orgrating 12, and the associated reflection angles, can be determinedusing known optical physics including Snell's Law, shown below:n _(in) sin θin=n _(out) sin θout  Eq. 9

where n_(in) is the refractive index of the first (input) medium, andn_(out) is the refractive index of the second (output) medium, and θinand θout are measured from a line 620 normal to an incident surface 622.

Referring to FIG. 36, if the value of n1 in the grating region 20 isgreater than the value of n2 in the non-grating region 18, the gratingregion 20 of the substrate 10 will act as a known optical waveguide forcertain wavelengths. In that case, the grating region 20 acts as a“core” along which light is guided and the outer region 18 acts as a“cladding” which helps confine or guide the light. Also, such awaveguide will have a known “numerical aperture” (θna) that will allowlight that is within the aperture θna to be directed or guided along thegrating axis 207 and reflected axially off the grating 12 and returnedand guided along the waveguide. In that case, the grating 12 willreflect light having the appropriate wavelengths equal to the pitches Λpresent in the grating 12 back along the region 20 (or core) of thewaveguide, and pass the remaining wavelengths of light as the light 632.Thus, having the grating region 20 act as an optical waveguide forwavelengths reflected by the grating 12 allows incident light that isnot aligned exactly with the grating axis 207 to be guided along andaligned with the grating 12 axis 207 for optimal grating reflection.

If an optical waveguide is used any standard waveguide may be used,e.g., a standard telecommunication single mode optical fiber (125 microndiameter or 80 micron diameter fiber with about a 8-10 micron diameter),or a larger diameter waveguide (greater than 0.5 mm diameter), such asis describe in U.S. patent application Ser. No. 09/455,868, filed Dec.6, 1999, entitled “Large Diameter Waveguide, Grating”. Further, any typeof optical waveguide may be used for the optical substrate 10, such as,a multi-mode, birefringent, polarization maintaining, polarizing,multi-core, multi-cladding, or microsturctured optical waveguide, or aflat or planar waveguide (where the waveguide is rectangular shaped), orother waveguides. Any other dimensions may be used for the waveguide ifdesired, provided they meet the functional and performance requirementsof the application taking into account the teachings herein.

Referring to FIG. 37, if the grating 12 extends across the entiredimension D of the substrate, the substrate 10 does not behave as awaveguide for the incident or reflected light and the incident light 24will be diffracted (or reflected) as indicated by lines 642, and thecodes detected as discussed hereinbefore for the end-incidence conditiondiscussed hereinbefore with FIG. 45, and the remaining light 640 passesstraight through.

Referring to FIG. 38, illustrations (a)-(c), in illustration (a), forthe end illumination condition, if a blazed or angled grating is used,as discussed hereinbefore, the input light 24 is coupled out of thesubstrate 10 at a known angle as shown by a line 650. Referring to FIG.38, illustration (b), alternatively, the input light 24 may be incidentfrom the side and, if the grating 12 has the appropriate blaze angle,the reflected light will exit from the end face 652 as indicated by aline 654. Referring to FIG. 38, illustration (c), the grating 12 mayhave a plurality of different pitch angles 660,662, which reflect theinput light 24 to different output angles as indicated by lines 664,666. This provides another level of multiplexing (spatially) additionalcodes, if desired.

The grating 12 may be impressed in the substrate 10 by any technique forwriting, impressed, embedded, imprinted, or otherwise forming adiffraction grating in the volume of or on a surface of a substrate 10.Examples of some known techniques are described in U.S. Pat. Nos.4,725,110 and 4,807,950, entitled “Method for Impressing Gratings WithinFiber Optics”, to Glenn et al; and U.S. Pat. No. 5,388,173, entitled“Method and Apparatus for Forming Aperiodic Gratings in Optical Fibers”,to Glenn, respectively, and U.S. Pat. No. 5,367,588, entitled “Method ofFabricating Bragg Gratings Using a Silica Glass Phase Grating Mask andMask Used by Same”, to Hill, and U.S. Pat. No. 3,916,182, entitled“Periodic Dielectric Waveguide Filter”, Dabby et al, and U.S. Pat. No.3,891,302, entitled “Method of Filtering Modes in Optical Waveguides”,to Dabby et al, which are all incorporated herein by reference to theextent necessary to understand the present invention.

Alternatively, instead of the grating 12 being impressed within thesubstrate material, the grating 12 may be partially or totally createdby etching or otherwise altering the outer surface geometry of thesubstrate to create a corrugated or varying surface geometry of thesubstrate, such as is described in U.S. Pat. No. 3,891,302, entitled“Method of Filtering Modes in Optical Waveguides”, to Dabby et al, whichis incorporated herein by reference to the extent necessary tounderstand the present invention, provided the resultant opticalrefractive profile for the desired code is created.

Further, alternatively, the grating 12 may be made by depositingdielectric layers onto the substrate, similar to the way a known thinfilm filter is created, so as to create the desired resultant opticalrefractive profile for the desired code.

FIGS. 39-50: Alternative Microbead Geometries

The substrate 10 (and/or the element 8) may have end-viewcross-sectional shapes other than circular, such as square, rectangular,elliptical, clam-shell, D-shaped, or other shapes, and may haveside-view sectional shapes other than rectangular, such as circular,square, elliptical, clam-shell, D-shaped, or other shapes. Also, 3Dgeometries other than a cylinder may be used, such as a sphere, a cube,a pyramid or any other 3D shape. Alternatively, the substrate 10 mayhave a geometry that is a combination of one or more of the foregoingshapes.

The shape of the element 8 and the size of the incident beam may be madeto minimize any end scatter off the end face(s) of the element 8, as isdiscussed herein and/or in the aforementioned patent application.Accordingly, to minimize such scatter, the incident beam 24 may be ovalshaped where the narrow portion of the oval is smaller than the diameterD1, and the long portion of the oval is smaller than the length L of theelement 8. Alternatively, the shape of the end faces may be rounded orother shapes or may be coated with an antireflective coating.

It should be understood that the size of any given dimension for theregion 20 of the grating 12 may be less than any corresponding dimensionof the substrate 10. For example, if the grating 12 has dimensions oflength Lg, depth Dg, and width Wg, and the substrate 12 has differentdimensions of length L, depth D, and width W, the dimensions of thegrating 12 may be less than that of the substrate 12. Thus, the grating12, may be embedded within or part of a much larger substrate 12. Also,the element 8 may be embedded or formed in or on a larger object foridentification of the object.

The dimensions, geometries, materials, and material properties of thesubstrate 10 are selected such that the desired optical and materialproperties are met for a given application. The resolution and range forthe optical codes are scalable by controlling these parameters asdiscussed herein and/or in the aforementioned patent application.

Referring to FIG. 39, the substrate 10 may have an outer coating 799,such as a polymer or other material that may be dissimilar to thematerial of the substrate 10, provided that the coating 799 on at leasta portion of the substrate, allows sufficient light to pass through thesubstrate for adequate optical detection of the code. The coating 799may be on any one or more sides of the substrate 10. Also, the coating799 may be a material that causes the element 8 to float or sink incertain fluids (liquid and/or gas) solutions.

Also, the substrate 10 may be made of a material that is less dense thancertain fluid (liquids and/or gas) solutions, thereby allowing theelements 8 to float or be buoyant or partially buoyant. Also, thesubstrate may be made of a porous material, such as controlled poreglass (CPG) or other porous material, which may also reduce the densityof the element 8 and may make the element 8 buoyant or partially-buoyantin certain fluids.

Referring to FIG. 40, the grating 12 is axially spatially invariant. Asa result, the substrate 10 with the grating 12 (shown as a longsubstrate 21) may be axially subdivided or cut into many separatesmaller substrates 30-36 and each substrate 30-36 will contain the samecode as the longer substrate 21 had before it was cut. The limit on thesize of the smaller substrates 30-36 is based on design and performancefactors discussed herein and/or in the aforementioned patentapplication.

Referring to FIG. 41, one purpose of the outer region 18 (or regionwithout the grating 12) of the substrate 10 is to provide mechanical orstructural support for the inner grating region 20. Accordingly, theentire substrate 10 may comprise the grating 12, if desired.Alternatively, the support portion may be completely or partiallybeneath, above, or along one or more sides of the grating region 20,such as in a planar geometry, or a D-shaped geometry, or othergeometries, as described herein and/or in the aforementioned patentapplication. The non-grating portion 18 of the substrate 10 may be usedfor other purposes as well, such as optical lensing effects or othereffects (discussed herein or in the aforementioned patent application).Also, the end faces of the substrate 10 need not be perpendicular to thesides or parallel to each other. However, for applications where theelements 8 are stacked end-to-end, the packing density may be optimizedif the end faces are perpendicular to the sides.

Referring to FIG. 42, illustrations (a)-(c), two or more substrates10,250, each having at least one grating therein, may be attachedtogether to form the element 8, e.g., by an adhesive, fusing or otherattachment techniques. In that case, the gratings 12,252 may have thesame or different codes.

Referring to FIG. 43, illustrations (a) and (b), the substrate 10 mayhave multiple regions 80,90 and one or more of these regions may havegratings in them. For example, there may be gratings 12,252 side-by-side(illustration (a)), or there may be gratings 82-88, spaced end-to-end(illustration (b)) in the substrate 10.

Referring to FIG. 44, the length L of the element 8 may be shorter thanits diameter D, thus, having a geometry such as a plug, puck, wafer,disc or plate.

Referring to FIG. 45 to facilitate proper alignment of the grating axiswith the angle θi of the input beam 24, the substrate 10 may have aplurality of the gratings 12 having the same codes written therein atnumerous different angular or rotational (or azimuthal) positions of thesubstrate 10. In particular, two gratings 550, 552, having axial gratingaxes 551, 553, respectively may have a common central (or pivot orrotational) point where the two axes 551,553 intersect. The angle θi ofthe incident light 24 is aligned properly with the grating 550 and isnot aligned with the grating 552, such that output light 555 isreflected off the grating 550 and light 557 passes through the grating550 as discussed herein. If the element 8 is rotated as shown by thearrows 559, the angle θi of incident light 24 will become alignedproperly with the grating 552 and not aligned with the grating 550 suchthat output light 555 is reflected off the grating 552 and light 557passes through the grating 552. When multiple gratings are located inthis rotational orientation, the bead may be rotated as indicated by aline 559 and there may be many angular positions that will providecorrect (or optimal) incident input angles θi to the grating. While thisexample shows a circular cross-section, this technique may be used withany shape cross-section.

Referring to FIG. 46, illustrations (a), (b), (c), (d), and (e) thesubstrate 10 may have one or more holes located within the substrate 10.In illustration (a), holes 560 may be located at various points alongall or a portion of the length of the substrate 10. The holes need notpass all the way through the substrate 10. Any number, size and spacingfor the holes 560 may be used if desired. In illustration (b), holes 572may be located very close together to form a honeycomb-like area of allor a portion of the cross-section. In illustration (c), one (or more)inner hole 566 may be located in the center of the substrate 10 oranywhere inside of where the grating region(s) 20 are located. The innerhole 566 may be coated with a reflective coating 573 to reflect light tofacilitate reading of one or more of the gratings 12 and/or to reflectlight diffracted off one or more of the gratings 12. The incident light24 may reflect off the grating 12 in the region 20 and then reflect offthe surface 573 to provide output light 577. Alternatively, the incidentlight 24 may reflect off the surface 573, then reflect off the grating12 and provide the output light 575. In that case the grating region 20may run axially or circumferentially 571 around the substrate 10. Inillustration (d), the holes 579 may be located circumferentially aroundthe grating region 20 or transversely across the substrate 10. Inillustration (e), the grating 12 may be located circumferentially aroundthe outside of the substrate 10, and there may be holes 574 inside thesubstrate 10.

Referring to FIG. 47, illustrations (a), (b), and (c), the substrate 10may have one or more protruding portions or teeth 570, 578,580 extendingradially and/or circumferentially from the substrate 10. Alternatively,the teeth 570, 578,580 may have any other desired shape.

Referring to FIG. 48, illustrations (a), (b), (c) a D-shaped substrate,a flat-sided substrate and an eye-shaped (or clam-shell or teardropshaped) substrate 10, respectively, are shown. Also, the grating region20 may have end cross-sectional shapes other than circular and may haveside cross-sectional shapes other than rectangular, such as any of thegeometries described herein for the substrate 10. For example, thegrating region 20 may have a oval cross-sectional shape as shown bydashed lines 581, which may be oriented in a desired direction,consistent with the teachings herein. Any other geometries for thesubstrate 10 or the grating region 20 may be used if desired, asdescribed herein.

Referring to FIG. 49, at least a portion of a side of the substrate 10may be coated with a reflective coating to allow incident light 510 tobe reflected back to the same side from which the incident light came,as indicated by reflected light 512.

Referring to FIG. 50, illustrations (a) and (b), alternatively, thesubstrate 10 can be electrically and/or magnetically polarized, by adopant or coating, which may be used to ease handling and/or alignmentor orientation of the substrate 10 and/or the grating 12, or used forother purposes. Alternatively, the bead may be coated with conductivematerial, e.g., metal coating on the inside of a holy substrate, ormetallic dopant inside the substrate. In these cases, such materials cancause the substrate 10 to align in an electric or magnetic field.Alternatively, the substrate can be doped with an element or compoundthat fluoresces or glows under appropriate illumination, e.g., a rareearth dopant, such as Erbium, or other rare earth dopant or fluorescentor luminescent molecule. In that case, such fluorescence or luminescencemay aid in locating and/or aligning substrates.

Further Alternative Embodiments for Groove Plates and Loading/UnloadingBeads

Referring to FIGS. 55 and 56, the bead cell, chamber, or cuvettes 900,920, respectively, may be segmented into regions each associated with adifferent reaction or used for a different identificationprocess/application. In particular, referring to FIG. 55, for a cellhaving circular grooves 1258, the cell may have a plurality of separatesections 902 which are physically separated from each other by barriers,904. In that case, the beads may be loaded through separate holes orports 906, which communicate only with an associated section 902. Thesections 902 may be mechanically isolated, so that the beads 8 placed ina given section 902 all remain in that section, and/or fluidicallyisolated, so that any fluid with the beads 8 placed in a given section902 remains in that section with no cross-over into any other section902.

Further, referring to FIG. 56, for a cell having straight grooves 205,the cell 940 may have a plurality of separate sections 942 which arephysically separated from each other by barriers, 944. In that case, thebeads may be loaded through separate holes or ports 946, whichcommunicate only with an associated section 942. The sections 942 may bemechanically isolated, so that the beads 8 placed in a given section 942all remain in that section, and/or fluidically isolated, so that anyfluid with the beads 8 placed in a given section 942 remains in thatsection with no cross-over into any other section 942.

Referring to FIG. 57, one example of a sectored cell 920 with straightgrooves 205 has a base groove plate 930, a spacer 932, and a cover 934.The groove plate may be made of fused silica, borosilicate glass, orplastic, acrylic, Zeonex made by Zeon Corp. or any other supportmaterial that is transparent or substantially transparent to desiredincident wavelength light or can be made of reflective by coating atransparent material or using a reflective material, such as silicon orother support material that reflects the desired wavelengths of incidentlight. Also, the groove plate 930 may be made of a material that hasminimal fluorescence to minimize background fluorescence in the desiredfluorescence wavelength range, for applications where fluorescence ofthe beads 8 is measured.

The base plate 930 has a substantially circular shape having a diameterof about 100 mm, with a mechanical alignment key or notch 952 about 32.5mm long, which may be used for mechanical alignment during waferfabrication of the groove plate 930. The thickness 948 is about 1 mm.The base plate 930 has the grooves 205 therein, which may be formed bydirect reactive ion etching (REI) of the glass base plate 930,photo-patterning with photoresist, photoresist and plating process, orany other process that provides the grooves 205 that meet therequirements for the application. The sectors 944 have a length 950 ofabout 50 mm. Also, one or more reference lines 948 (or fiducials) may beprovided for reader head alignment with the grooves 205. The length 940of each grooved section or sector 944 is about 7 mm and the space 946between each section 944 is about 2 mm. The grooves 205 are about 34microns by 24 microns deep and have about a 55 micron pitch spacing. Fora 7 mm long groove, each groove 205 would hold about 28 cylindricallyshaped beads 8 each bead 8 having a dimension of about 30 microns indiameter and 250 microns in length. The sectors 944 having a length ofabout 50 mm, may have about 900 grooves and hold a total capacity about25,200 beads 8. While the number of physically separated sectors 944 inthe cell 938 shown is eight, any number of sectors may be used ifdesired.

Referring to FIG. 58, the grooves 205 have a depth of about 22 to 24microns, and have a top width of about 34 microns, and a base width 953of about 30 microns for θg=5 deg., and a spacing pitch of about 55microns, for a bead 8 having a diameter D1 of about 28 micons. The sidewalls 958 may have an angle θg of about 0 to 10 degrees. Other anglesmay be used, depending on the application, e.g., whether the beads willbe removed from the plate and how they will be removed.

For example, referring to FIG. 58, with the angle θg is between 0 andabout 10 degrees the beads may be flushed or washed out of the grooves205 with fluid flow transversely across the top of the grooves 205,using a fluid flow rate of about 3 to 6 ml/second cleans out the beads.The flush may be done with dionized water, regular water, saline,detergent with water, or other liquid. Using a detergent reduces theviscosity and surface tension so beads do not stick to the surface ofthe cell. The angle θg may be greater than 10 degrees if desired,depending on certain design parameters, including, flush flow rate,groove-to-groove separation, and groove depth. Alternatively, if theangle θg is less than 0 deg., the beads will be more likely to stay inthe grooves 205.

Other dimensions and geometries for the groove plate 930, grooves 205,spacer 932, and cover 934 and/or for any features or characteristicsthereof may be used if desired.

Loading/Moving Beads Using Pressure Wave/Vibrations

The present invention, which is predicated on two observations,eliminates the need for mechanically distributing beads. The firstobservation is that small particles are easily moved by a fluid stream,and the orientation of cylindrical particles is generally with the longaxis of the particle perpendicular to the direction of the flow. And thesecond is that particles in a liquid can be moved in a particulardirection by a temporally asymmetric oscillatory flow. Regarding thelater, it was observed that when an oscillatory flow was used in aclosed fluidic cell containing cylindrical glass particles, whereby therate of the outgoing wave was higher than the return wave, the particleswould acquire a net displacement in the direction of the outgoing wave.When the flow rates were reversed, i.e. when the outgoing wave wasslower than the return wave, the particles moved inward. Again it wasobserved that the particles would tend to orient perpendicular to thedirection of the pressure wave.

This behavior was first observed using a closed fluidic cell in theshape of a round disk with a floor and ceiling spaced by approximately500 micron. The cell was entirely closed except for a hole in the centerof the top, which allowed the particles (400×40 um cylindrical glass“beads”) to be inserted into the center of the cell. An asymmetric flowwas established by tapping the bottom of the cell with a blunt object. Atime sequence is shown FIG. 1(a-f), illustrating how the particles forma ring shaped pattern and how the size of the ring increased, indicatingthat the particles were moving outward, after a series of pulses wereapplied in one direction. FIG. 1 (g−1), illustrate how the size of thering decreased after the direction of the pulses was reversed. Insubsequent experiments, oscillatory flow was established by couplingfluid through the open port in the top of the cell. The general behaviorof the cell was the same in either case. By applying rapid pressurepulses, coupled through a flexible tube inserted into the center hole,and allowing the waves to slowly return, beads were made to moveoutward, thereby forming the familiar circular shape. The radius of thecircle depended on such things as: the number of pulses, the amplitudeof the pulses, the separation between the floor and the ceiling, thesize of the beads and the geometry of the cell. An important feature ofthe cell was an air buffer around the perimeter of the cell to allow thefluid a place to move, since the fluid itself is non-compressible, theair gap acted as a pneumatic spring. Another important feature was thespace between the floor and ceiling. It was important to maintain asmall gap (<500 um) between the floor and the ceiling to keep thevelocity of the fluid in the cell high enough to move the particles.

Other experiments relating to the general behavior of fluidic-inducedparticle movement include placing cylindrical beads on the bottom of aan open vessel such as a beaker, then moving the beads by introducingthe tip of a syringe into the pile of beads and blowing the liquid outthrough the tip. In this experiment, the beads all moved radially awayfrom the tip, leaving behind a region void of all beads. Again, it wasobserved that the beads tended to generally align parallel to the wavefront. FIG. 11 shows a schematic of a concept that uses two suchflow-generating tips. The flow from the tips can be operated such thatthey oppose each other, thus acting to push the beads into the regionhalf way between the tips. Or they can be operated in a push-pullfashion whereby the beads tend to move toward one tip or the other. Asynthetic circular force field can be generated by rotating the platewhile operating the tips in either of the previously mentioned methods.

An of the invention involves combining the ability to transport beadsacross the floor of a substrate using either continuous fluid flow or atype of asymmetric oscillatory flow, with the technology for trappingbeads, such as the previously described groove plate. This would enablea highly efficient assembly of beads with precise orientations in thesmallest possible area. With respect to reduced operating cost and highthroughput, all three of these attributes are important elements of acommercial encoded particle reader. FIG. 70 shows a schematic of aconcept that incorporates a closed liquid cell and the elements requiredto load the cell efficiently. Key elements of the method include: aclosed cell including a top and a bottom, the bottom contains a platewith grooves for aligning beads, both the top and the bottom aretransparent, the top has an opening in the center for loading beads andfor coupling a pressure generating device such as a bellows or a tube,and finally a region of trapped air around the perimeter adjacent to andin contact with the fluid in the cell. The loading operation consistsof: filling the cell with a liquid such as water, spinning the cell toremove the air bubbles, dispensing beads through the center hole in thelid, applying a pulsating flow such that the rate of the outward goingpulse is higher the return pulse. This will tend to move the pile ofbeads away from the center of the cell. As the beads move outward theypopulate the grooves. The direction of the pulsation can be reversed tomove the pile of beads back toward the center to enhance the probabilitythat the grooves are fully populated before allowing beads to move outto a larger radius. By moving the beads in and out it should be possibleto fully populate the inner most grooves, thus maximizing the overallloading density. It may further be desirable to include an azimuthal (orcircumferential) agitation or vibration to stimulate the beads to movealong the channels of the grooves once they have fallen in, therebyenhancing the probability that an open space is created to allow roomfor additional beads to fall into the groove.

Also see FIGS. 77-79 for “puffing” (pressure pulses) done with straightgrooves with the actuator on one end.

Unloading Beads

Referring to FIG. 80-81, the beads may be unloaded by flushing withfluid as shown and discussed herein before.

Cylindrical Groove Platform

Alternatively, referring to FIGS. 62-65, the groove plate may becylindrical shaped. In that case, the grooves 205 may run along thelongitudinal axis as shown in FIGS. 62,63 or circumferentially as shownin FIGS. 64,65. The grooves 205 may be oriented in any other directionalong the cylindrical groove if desired. Also, the grooves 205 may belocated on the outside of the cylinder as shown in FIGS. 62,64.Alternatively, the grooves 205 may be located on the inside of thecylinder as shown in FIGS. 63,65. When the grooves 205 are located onthe inside, the cylinder may be spun about the longitudinal axis tolocate the beads 8 within the grooves 205. The orientation of thelongitudinal axis of the cylinder may be such that the longitudinal axisis vertical or horizontal or at any other desired angle.

Referring to FIG. 89, an apparatus for transporting microbeads for thepresent invention includes a container or well 400 with a sealed lid 410having microbeads 8 and liquid 412 therein and a second container 402having a sealed lid 411 with liquid 412 therein. Two tubes 406,408penetrate the lid 410, the first tube 406 connects the first container400 to a pump 416 and the second tube 408 connects the first containerto the second container 402 for receiving the beads from the firstcontainer 200. The first container 400 is filled with a liquid 412, andone end or tip 414 of the first tube 406 is in the liquid 412 apredetermined distance into the container 400, e.g., 20% to 50% of thefull depth of the container. A tip 409 of the tube 408 is mountedsubstantially flush with the bottom surface of the lid 410. The pump 416pumps liquid 412 from a reservoir 405 into the tube 406 to the firstcontainer 400. When the pump 416 pumps the liquid 412 into the container400, the beads 8 are agitated as indicated by the lines 418. The liquid412 and the beads 8 exit the container 400 through the tube 408 asindicated by a line 420 and are emptied into the container 402. Theliquid 412 enters the container 402 and exits the container 402 througha filter 424 then through a tube 428, as indicated by a line 426. Thefilter 424 prevents the beads 8 from exiting the container 402. Theliquid 412 that exits the container 402 is dispensed via the tube 428into a waste pan or container 430.

Instead of the pump 416 being connected to the tube 406, a vacuum pump432 may draw a vacuum on the tube 428. In that case the tube 406 wouldbe open ended. We have found that this technique transports all thebeads 8 from the container 400 to the second container 402.

The tip 409 of the tube 408 may be placed further into the container(i.e., not flush with inner surface of the lid 410), if desired. In thatcase, some air may be pumped along the tube 408 with the liquid and thebeads 8. If air exists at the top of the container 400, the beads 8 maystick to the wall or inner surface of the lid 410 and not be transportedto the other container 402.

Referring to FIG. 90, instead of the second container 402 being a sealedcontainer, it may be an open container. In that case, the container 402should have sufficient volume to receive the fluid from the pump 416.

Referring to FIGS. 90 and 91, instead of having the filter 424 on theexit port of the sealed container 402, the container may have a volumethat is large enough such that when the beads 8 enter from the tube 408,they stay substantially near the bottom of the container 402 and do notget sucked out of the tube 428 to the waste container 430. Thistechnique for transporting the beads 8 may be referred to as the“telegraph” technique.

The seal between the lids 410,411 and the containers 400, 402, may beany type of seal that retains the liquid inside the container, e.g., aradial seal/inner surface seal on the inside wall of the container, atop surface/axial seal to the top surface of the container, or any otherseal that will perform the function required.

We have found that a flow rate of 1.0 to 2.0 ml/sec., with a tube innerdiameter of 0.031 to 0.063 inches, and a total transport time of about0.73 seconds will transport all the beads from a well of a 96 well plateto a bead reader cell, such as that described in copending U.S. patentapplication Ser. No. (CyVera Docket No. CV-0082 PR). In that case, thefirst container 400 would be an individual well in the well plate, andthe second container 402 would be the reader cell.

Also, this can be automated such that the lid 410 is a probe head whichcomes down on top of the well to create a seal on the well. The probewould contain the two tubes 406, 406, the tube 406 would be an aspiratetube and the tube 408 would be a dispense tube for dispensing ortransporting the beads 8 from the first container 400 to the secondcontainer 402. As discussed herein, the system can operate underpressure or a vacuum. For a system operating under pressure, the liquid412 is driven into the dispense line, pressurizing the well and sendingthe fluid out of the aspirate tube 408. This permits use of drivepressure greater than 1 atm. However, there is a risk that fluid (andpossibly beads) will leak out of the well if the lid seal fails. In avacuum configuration, the aspirate tube 408 is connected to negativepressure, and drive pressure is limited to 1 atmosphere. However, inthat case, if a seal fails, air leaks into the system instead of liquid(and possibly beads) leaking out.

Referring to FIG. 92, a similar configuration to that shown in FIG. 91,using a syringe pump.

Referring to FIGS. 93-97, various alternative configurations for pullingor pushing the beads 8 out of a well 440 having a sealed lid 446,through a tube 442, to a larger diameter holding area 440, using asyringe pump 448. In each case, once the beads 8 are in the holding area440, the lid 446 is removed from the container 440 and placed in thetarget or destination well or container (not shown).

In particular, two tips penetrate the upper seal on the container asdiscussed hereinbefore, with one tip connected to a syringe pump and theother connected to a reservoir. When the syringe pump is aspirated,fluid will be pulled from the reservoir through the second tip. Thefluid is thus dispensed from the second tip, agitating the slurry, andaspirated by the first tip. In this way, an arbitrary volume of fluidcan be dispensed and aspirated using only a single pump, withoutoverflowing the well or prematurely emptying the well of fluid. Thedispensing head is then moved to the new location desired. To dispensethe beads, flow is reversed. The flow rate is set lower to avoidre-aspirating the beads into the reservoir. Also, in general, the volumecan be set much lower to simply dispense the beads into a new well. Thevolume can be set the same, however, to refill the reservoir to theoriginal volume. Alternatively, the actuation direction can be reversed.The second tip can be connected to a pump, while the first tip isconnected to the reservoir. When fluid is dispensed under pressurethrough the second tip, fluid will flow up through the first tip,providing an effective aspiration. Re-dispense then involves aspirationthrough the second tip.

Referring to FIG. 98, a fluidic circuit for loading beads 8 from wells400 in a known multi-well plate (e.g., a 96 well plate having 8 rows and12 columns) to a multi-segmented bead cell/chamber, such as is discussedin copending U.S. patent applications Ser. No. (CyVera Docket No.CV-0082 PR) and copending U.S. patent applications Ser. No. (CyVeraDocket No. CV-0086 PR) is shown. This system uses an air pump to createa vacuum to pull the beads into the cell 402 (second container) from thewells 400 (first container).

In particular, source fluid is contained in reagent bottles. A bottle isselected by opening the valve which leads by a tubing connection throughthe bottle cap to the desired bottle. Three bottles are shown, actuatedby valves V1, V2 and V3. Additional bottles could be added, each with acompanion valve. All valves are electrically operated solenoid valves,such as clean valves sold by Takasago Corp. Valve V6 is ideally a tubingpinch type valve for reliability as beads may damage a conventionalsolenoid valve.

The prime mover in this embodiment is an air pump, such as that made byBoxer Corp., which creates a vacuum condition in a pressure vessel thatacts as a vacuum trap. Fluid is then pulled into this container whenvalve V6 is open.

Alternatively, a liquid pump can serve as the prime mover. In this case,a filter should be placed in front of the pump to block beads fromentering the pump. If a liquid pump is used, the pressure vessel isunnecessary as an unsealed waste container can be used. Alternatively, asyringe pump, such as that sold by Kloehn Inc., can serve as the primemover (as shown in FIG. 94). In this case, a filter should be placed infront of the syringe pump to block beads from entering the syringe. Athree way valve must be used with the syringe pump so that after fillingthe syringe, fluid the valve can be switched to then dump fluid towaste. If a syringe pump is used, the pressure vessel is unnecessary asan unsealed waste container can be used.

Referring to the valve state table shown as Table 1 below, to describethe process of filling the cell, begin with a null state of all valvesclosed and the pump off. The pump is turned on to stabilize a vacuumcondition in the pressure vessel. One of valves V1, V2 or V3 is opened.Valve V4 is opened to direct fluid into the cell. Valve V6 is opened,thereby pulling fluid from the reagent bottles, through the cell andinto the pressure vessel. Valve V6 controls the fill cycle time and isheld open for a specified length of time, e.g., 1 second, calibrated topull the desired volume of fluid through the cell.

A bubble sensor, such as that made by Introtek, may be used to aid infilling the cell with fluid, by ensuring that the fluid line is free ofair before ending the fill cycle. The bubble sensor may also be used todetect if air is being pulled into the cell or system by an improperlyseated lid 410 or other air leak. An optional bubble sensor may also beused near the reagent bottles to detect when one of the reagent bottlesare empty. Alternatively, a level sensor, such as that made by TheMadison Company, in each reagent bottle may be used instead of thebubble sensor to detect empty bottles. Also, another level sensor may beused to in the waste container to detect a full containter.

Continuing the cycle, with the pump on, valve V6 closed and V4 open, tomove beads from the well plate into the cell, valve V4 is closed andeither valve V5 or valve V7 is opened. Valve V7 is only used if thesingle well to 8 output divider is intended to be used. Valve V6 isopened, thereby pulling fluid from the reagent bottles, into the sealedwell plate, out the well plate into the cell, pushing fluid out of thecell into the pressure vessel. Beads are pulled along with the fluidfrom the well plate into the cell. While excess fluid exits the cell,the beads remain due to the manifold design within the cell.

To flush beads from the cell, the process of filling the cell isrepeated. Cycle time is set longer, e.g., 2 to 5 seconds, for flush thanfor filling the cell, as several volume changes are desired to cleanfluid and beads from the cell. As the flush volume is several timesgreater than the volume held within the cell, and the fluid velocity ishigh, the beads are propelled out of the cell, pass through valve V6 andenter the pressure vessel. A filter in the pressure vessel can be usedto capture the beads. Standard household or industrial water filterhousing makes an excellent pressure vessel, as does bag filter housing,such as the Giant Bag Housings by MetPro Corporation, Keystone FilterDivision. TABLE 1 Valve Number V1 V2 V3 V4 V5 V6 V7 Fill Cell With FluidOPEN OFF OFF OPEN OFF OPEN OFF Transfer Beads from 1 OFF OPEN OFF OFFOPEN OPEN OFF Well to 1 Sector in Cell Transfer Beads from 1 OFF OPENOFF OFF OFF OPEN OPEN Well to 8 Sectors in Cell Load Beads OFF OFF OFFOFF OPEN OFF OFF Flush Reagent #1 OPEN OFF OFF OPEN OFF OPEN OFF FlushReagent #2 OFF OPEN OFF OPEN OFF OPEN OFF Flush Reagent #3 OFF OFF OPENOPEN OFF OPEN OFFIn Table 1, Off = fluid flow is blocked; Open = valve passes fluid flow.

Referring to FIG. 99, an alternative fluidic circuit for loading beads 8from wells 400 in a known multi-well plate (e.g., a 96 well plate having8 rows and 12 columns) to a multi-segmented bead cell/chamber is shown.The bead cell is similar to that described in copending U.S. patentapplications Ser. No. (CyVera Docket No. CV-0082 PR) and/or copendingU.S. patent applications Ser. No. (CyVera Docket No. CV-0086 PR). Thissystem uses an air pump to create a vacuum to pull the beads into thecell 402 (second container) from the wells 400 (first container). Thissystem also uses a 3-way valve to route the various fluids into the cell402 or into the wells 400.

Referring to FIG. 100, an alternative fluidic circuit for loading beads8 from wells 400 in a known multi-well plate (e.g., a 96 well platehaving 8 rows and 12 columns) to a multi-segmented bead cell/chamber 402is shown. The bead cell is similar to that described in copending U.S.patent applications Ser. No. (CyVera Docket No. CV-0082 PR) and/orcopending U.S. patent applications Ser. No. (CyVera Docket No. CV-0086PR). This system uses an air pump to create a vacuum to pull the beadsinto the cell 402 (second container) from the wells 400 (firstcontainer). This system also uses a cell having a filter or frit asdescribed in the aforementioned patent application to help collect thebeads at the entry of the cell prior to distributing the beads acrossthe cell for reading.

Referring to FIGS. 101-102, an example of an O-ring sealed lid 410 thatfits on top of the well (or first container) 400 that would contain thebeads 8. FIG. 102 shows a head having eight lids 410, one for each wellof an eight row well plate and the tubes 406,408, as well as a housingto which they are all mounted.

Referring to FIG. 110, a cross-section of a head having 8 lids 410,engaged with eight wells, and also showing a housing and springs and thetubes 406, 408.

Referring to FIG. 103, a fluidic circuit for loading beads 8 from wells400 in a known multi-well plate (e.g., a 96 well plate having 8 rows and12 columns) to a multi-segmented bead cell/chamber 402, similar to thatdescribed herein with FIGS. 98 and 99, except that this system uses apipetting technique instead of a “telegraph” technique for moving thebeads. In that case, the pipette is placed in a well 400 and beads areextracted into the pipette tip. Then the pipette tip is moved andinserted into a pipette port on the cell 402. Also, this embodiment usesa 3-way valve for flow management.

Referring to FIGS. 108-109, instead of moving beads from one well to oneof the sectored cells in the cell 402, a 1 to 8 flow manifold may beused to distribute beads from one well to eight separated sectors in thecell. FIG. 109 (a) is a perspective view and FIG. 109 (b) is a sidecross-section view of the 1 to 8 fluid manifold. This 1 to 8 manifold isalso shown as one option in the fluidics schematic if FIG. 98. The 1 to8 manifold may be used to move fluid (with or without beads) from onewell or port to 8 wells or port or used in as an 8 to 1 manifold to movefluid (with our without beads) from 8 wells or ports to 1 well or port.

Referring to FIGS. 104-106, one technique for pipetting a predeterminednumber of the beads 8 from a well 462 is as follows:

1. Start with a highly accurate estimate of the total number of beads 8in a large population in a separate container (not shown). This can bedone by aspirating a certain volume of beads 8 and knowing the packingdensity is around 40%. N=volume of beads (ul)×40%/volume per bead(ul/bead).

2. Dispense the beads 8 into a known volume of buffer solution, e.g.,SSC, SDS, or any other buffer solution or desired fluid in a vial orwell 462.

3. Calculate the concentration of the beads 8.

4. Agitate the mixture of the beads 8 in the vial 462 by repeatably andrapidly cycling the pipette 464 in the buffer solution 462, therebycausing the beads 8 to mix and suspend substantially “homogeneously” inthe solution 462. The agitation volume should be about 2-10% of thetotal volume and the rate of agitation should be fast enough to suspendthe beads in solution. Also, it was found that in order to generate goodfluid currents and, thus, good bead mixing/suspension, the pipette tipshould be placed away from the center of the well 460, and near to theside wall if possible. Note that the pipette tip should be inserted intothe liquid 462 such that the pipette tip is near the top of the liquidwhen the fluid is fully aspirated. Therefore, as the liquid leveldecreases from successive aspirations, the tip will need to be placeddeeper into the vial each time a new group of beads is removed. Also,the size of the opening in the pipette tip opening/orifice determinesthe velocity of the mixing currents for a given agitation volume andrate. For example, a larger orifice will result in lower velocities forthe same rate and volume. We have found that a tip with a small orifice(<about 600 um) works well for the 28×250 micron beads, solution andvolume used. However, the tip orifice should not be so small (<about 300um) that the flow through the tip for the pressure generated anddecreased to the point where the velocities are too low to generate goodmixing and suspension of the beads.

5. When the beads 8 are substantially “homogeneously” mixed andsuspended in the liquid, then the next (final) aspiration of beads 8should determine how many of the beads 8 are drawn from the vial 460.The number of beads 8 drawn=concentration (beads/ul)×aspiration volume(ul).

6. Dispensing the beads 8 into the target well 468 should include abrief time delay of about 1-3 sec to allow the beads 8 to fall to thebottom of the pipette tip before they are completely dispensed into thetarget well 468.

In particular, referring to FIG. 104, for example, starting with thevial 460 with about 1000 beads having a diameter of about 28 microns anda length of about 250 microns, in about 1000 microliters of known buffersolution 462, e.g., SCC,SDS. First insert a pipette tip 464 into theliquid 462 such that the pipette tip is near the top of the liquid whenthe fluid is fully aspirated. Then, holding the pipette tipsubstantially still, aspirate/dispense about 4 times with about 150microliters over a period of about 4 seconds; however other times may beused provided sufficient bead mixing and suspension is achieved. Then,draw a final aspiration of about 50 microliters. Then, transfer thepipette to a target well 468 and wait about 1-3 seconds to allow thebeads to settle to the end of the pipette tip, then dispense the beads 8into the well 468. The size of the opening in the pipette tip was 400microns (0.4 mm) and the size of the well 460 was about 1000 micronliters, and the pipette tip was placed about mid way between the centerof the well 460 and the side wall.

Referring to FIG. 105, a picture of a Hamilton Syringe Pump syringe pumpused to pipette beads with the present invention is shown. The pumphaving a storage buffer of 1×SSC, 0.1% SDS, and using 200 microliterultrafine points (VWR) pipette tips.

Referring to FIG. 106, a graph of syringe pump bead pipetting results isshown using the process described herein. For 36 tests, the averagenumber of beads removed each time was 18, with a bead diameter of about28 microns and length of 250 microns, agitation volume of 150microliters, final aspiration volume of 27 microliters, and a startingbead concentration of 0.68 beads per microliter.

Referring to FIG. 107, a diagram of how a kitting process may beperformed with the present invention. A plurality of containers or wells500-504 are provided, each well having beads with a specific code. Forexample, the well 500 has beads 8 with a code of 345, as shown by thedigital representation image 506, the well 502 has beads 8 having thecode of 8197, as shown by the digital representation image 508, and thewell 504 has beads 8 having the code of 15606, as shown by the digitalrepresentation image 510. The plurality of wells 500-504 having thebeads 8 can create Multiplex Bead Kits 1 through N, each Kit in aseparate container 516-520, and each Kit having a predetermined numberof any one or more of the codes in the wells 500-504. The beads 8 may betransported from the wells 500-504 using the any embodiment of thepresent invention or using any technique now known or later developed tomove a predetermined number the beads 8 into the containers 516-520 forthe Kits. The predetermined number of beads 8 of each code in each kitmay have a tolerance, e.g., +/−10 beads. Other bead kit tolerances maybe used depending on the application. Referring to FIGS. 111-114, amethod for making a “kit” consisting of N unique codes, where N mayrange from 1 to 5000, represented by M replicates (beads), where M mayrange from 5-100, can be accomplished by a two step process, consistingof transferring a small number (M) of beads from a vial or wellcontaining beads of all the same code to a target well or vial, then,combining the small number of beads representing each code in the kit toa single vial or well, thus forming the “kit”. The first step,transferring M beads from the source, could be performed in a 48,96 or384 well format using the pipetting approach previously described, orfrom an arbitrary configuration of individual vials. It is recognizedthat this can be done in parallel with a conventional multi-headpipetting machine such as those found in many laboratories. The secondstep of combining individual sets of N codes together to form the final“kit” may be accomplished by either dispensing the individual sets intoa funnel-like device where the beads are flushed into a single well orvial containing a filter bottom such that copious amounts of fluid maybe used to sufficiently flush all the beads through the funnel, leavingsubstantially no beads behind. Another approach, which accomplishes thecombining effect, is to “telegraph” (previously described) the beadsrepresenting individual codes into a single vial or well all at once.This process is very fast and highly efficient in terms of transferringall the beads from the source to the destination. This two-step processwould enable “kits” to be made with an arbitrary number of codes andrepresented by an arbitrary number of beads per code, in a rapid andefficient manner.

Referring to FIG. 115 shows a perspective view of the 8 sector bead cellhaving 8 input tubes 408 which transport beads and fluid from 8 cells to8 corresponding sectors of the bead cell. It also shows a 1 to 8 flowmanifold which takes in fluid and distributes it to 8 sectors in thecell.

In particular, FIG. 111 shows a multi-well plate having beads which arepipetted individually to another multi-well plate which are thentelegraphed as a group of wells (as described herein) to a filter wellKit container. FIG. 112 shows a multi-well plate having beads which arepipetted as a predetermined group or individual pipette tips to anothermulti-well plate, which are then telegraphed as a group of wells (asdescribed herein) to a filter well Kit container. FIG. 113 shows an 8 to1 manifold for receiving 8 pipette tips which will simultaneouslydispense fluid and beads into the manifold and the manifold combines thereceived fluid and beads to a single output port which dispenses thefluid and beads into the Kit container having a filter on the bottom tocatch the beads. The beads are then transferred to a final kittingcontainer. FIG. 114 shows a pipetting machine having a flush port forflushing fluid through the pipette tip, which can be used after thebeads 8 are dispensed into the manifold shown in FIG. 113, or wheneverflushing with a fluid is needed. In each of the above cases, the Kitcontainer may have a filter on the bottom to catch the beads and allowthe fluid to exit. The beads are then transferred to a final kittingcontainer. Alternatively, the container may be large enough to hold thefluid and the beads and then the beads and a portion of the fluid may betransported (e.g., by the telegraph method described herein) to asmaller kit container if needed.

An Alternative Embodiment of the Fluidic Subsystem

FIGS. 118-133 show an alternative embodiment of the present invention.In summary, FIGS. 118-121 show the basic achitecture and governingdesign principles; FIGS. 122 a to 125 show steps of the overall methodand the sequencing thereof; FIG. 126 shows details related to a grooveplate design; FIGS. 127 a-d show basic experiments; and FIGS. 128-133show more detailed diagrams of components of the basic architecture.

Consistent with that discussed above, the microbead platform willperform biological assays on beads by attaching a type of biomolecule tothe beads then placing the beads in a vessel containing sample material,which will react in varying degrees to the biomolecules. The extent towhich the sample reacts is determined by measuring the intensity of afluorescent tag molecule, and the indentity of the fluorescent beads isdetermined by reading its holographic code. Both fluorescence and codedetection methods place requirements on how beads are oriented relativeto the interrogation lasers and collection optics. The purpose of thefluidic sub-system is to manage all fluid and bead manipulationactivities entailed in the interrogation process. These include movementof beads from the microtiter plate to the cell, alignment of beads inthe cell and finally removal of beads from the cell so that the nextbatch can be interrogated. The fluidic system must also provide a meansby which it can be cleaned of all biological and chemical contamination.

The following 6 steps describe the basic functions of the reader from afluidic point of view:

-   -   1) Prime Cell    -   2) Transfer Beads    -   3) Load beads into grooves    -   4) Scan beads    -   5) Flush    -   6) Clean cell (after N cycles)

Steps 2-5 are performed every cycle on 8 wells at a time on an 8×12 wellplate. Therefore a full 96 well plate requires 12 cycles to complete.Step 1 is performed on start up and whenever the fluidic system eitheraccumulates too much air or to remove persistent beads from grooves.Likewise, step 6 is performed when an unacceptable level of fluorescentcontamination has accumulated.

FIG. 119 shows the governing design principles.

FIG. 120 shows a diagram of the fluidic system process flow includingthe 6 basic steps.

FIG. 121 shows the fluidic system including the architecture generallyindicated as 1000 and major subsystems, including a transfer tube orprobe assembly 1012, a well plate 1012, a cell or cell assembly 1008, apuffer tube or puffer tube assembly 1006 and syringe pump 1001,consistent with that shown in detail below in FIG. 118.

Fluidics Architecture

FIG. 118 shows in detail the basic fluidics architecture generallyindicated as 1000 for performing the 6 steps of the alternativeembodiment of the present invention, and includes the following elementsand functionality as set forth below:

The Syringe pump 1001 aspirates or dispenses from selected valveposition.

The Rotary valve 1002 rotates to select from 5 positions—Output to cell,Reagent 1, Reagent 2, Reagent 3, Dispense Excess/Aspirate Air.

The Tube Assembly, Output to Cell 1003 is fluoropolymer tubing to allowfluid to flow from syringe 1001 to the manifold leading to cell chimney1007.

The Manifold (1×8) 1004 divides flow from 1 input line to 8 output linesleading to cell.

Check valves 1005: Check valves are connected to each of the 8 lines inthe manifold. The check valves prevent fluid from siphoning betweenlanes of the cell. Without the check valves, a small height imbalance ofthe fluid in one probe versus another, could cause the siphoning offluid out of the line carrying the shorter fluid path. This could thenlead to a chain reaction where all the fluid siphons out of the cell.

Puffer tube assemblies 1006: The puffer tubes are constructed ofsilicone tubing with inner diameter 3/16 inch and outer diameter 5/16inch. Compression of the tubing with the puffer block displaces fluid.The check valves prevent fluid from flowing back to the pump. Also, evenwithout the check valves, the syringe is a stiff system which wouldprevent flow in this direction. Therefore all flow from compressionmoves out through the cell and out the probes. Release of the tubingthen aspirates fluid through the probes back into the cell. The siliconetubes were selected for their elasticity and low compression set.

The Chimney 1007—The chimney 1007 is a molded part that is critical tothe transfer of beads into the cell and loading of beads into grooves.The chimney 1007 terminates in the cell in a narrow line shaped nozzlewhich we call the line port. This shape provides a relatively flat flowvelocity profile across the width of the alley. The narrowness of theport (generally less than 2 bead lengths at the narrowest portion),prevents large slow moving eddy regions when beads turn the corner fromchimney to cell. Also, the spacer is aligned with the back of the nozzleto prevent significant dead zones of flow. Bead loading into groovestakes place at flow velocities that are in the laminar regime away fromthe immediate vicinity of the grooves. The chimney 1007 expands into awider region. The height of the chimney and width of the expanded regionwere designed to limit the height to which beads rise in the chimneyduring transfer. By limiting this height, beads are not aspirated out ofthe chimney, which would lead to cross-contamination in later cycles.

Cell Assembly is labelled 1008.

Top Plate 1008 a: The top plate is the top optical window of the cellsandwich. It contains a row of 8 holes for attachment of the chimney tothe 8 lanes of the cell. At the opposite end of the alleys, it containsa row of 8 holes for attachment of the bead ports.

Spacer 1008 b—The spacer maintains a gap between the groove plate andtop window. It also seals and separates the 8 alleys from each other andfrom the outside world. The spacer is made of a silicone gasket. Thegasket is attached to the two glass plates under compression and heat tocreate a seal. The spacer thickness is 0.015 inch or 380 microns. Thisthickness appears to be near an optimum value for balancing competingneeds. On one hand, the thinner the gap the higher the velocity near thegrooves, this aids in bead loading with the puffer and in bead removal.On the other hand, if the gap is too small, bubbles are not effectivelycleared from the cell. Note that other materials could serve as agasket.

Groove Plate 1008 c—the groove plate arranges the beads in an orderlyfashion to be read by the reader optics. The groove plate is made offused silica and is produced by an RIE (reactive ion etch) process.Fused silica is used for its low fluorescence, permitting bettersensitivity to low fluorescence signals. Several other processes havebeen explored for constructing a groove plate.

Bead Entrance/Exit Ports 1009—The bead tubes from the probes terminatein this port. The gasket taper to a rounded cone, with the port at theapex. The goal is to minimize dead volume, so that beads maintainmomentum as they enter and exit the cell through the port. Each alleyhas a fluidically isolated port.

Bead Tubes, fluoropolymer 1010—The bead tubes carry beads into the cellthrough the transfer process, and carry them out of the cell during theflush process. Fluoropolymer tubing is used for inertness, to minimizefriction and reduce bubble adhesion.

Probe Assemblies 1011—The probes are connected and integral with thebead tubes. The probes enter the well plate for transferring beads intothe cell. The probes are designed to withstand “bottoming out” in thewell plate and are spring loaded.

Well Plate 1012

Water Tube Assembly 1013

-   -   Tube Assembly, External, Water 1013 a

Buffer Tube Assembly 1014

-   -   Tube Assembly, External, Buffer 1014 a

Alcohol Tube Assembly 1015

-   -   Tube Assembly, External, Alcohol 1015 a

Air & Fluid Excess Tube Assembly 1016

Tee 1016 a

Air Inlet to Valve Check Valve 1016 b-On syringe aspirate from thisvalve position, allows air to enter the syringe. The check valve blocksflow in the dispense direction

Air Inlet to Valve Check Valve 1016 c—This check valve allows flow inthe dispense direction to waste, but blocks return flow, to preventaspiration from waste.

Waste Drain Tube Assembly 1017

-   -   Tube Assembly, External, Waste Drain 1017 a

Panel Connections 1018—Field connections for the customer. Luer locksare preferred.

Reagent Bottles 1019

Bottle Caps 1019 a

Tube Connections 1019 b

Straws 1019 c

Filters 1019 d

Level Switches 1020—Sense reagent bottles empty below a set point orwaste bottle full over a maximum level.

Waste Bottle 1021

Waste/Wash Tray 1022—Divided into two sections—one for dumping wastefluid and beads, and a section for washing the probe tips. Spillage fromthe wash overflows into waste. Fluid may be pumped using the auxiliarypump into the wash section to augment cleaning and to add bleach.

Wash subsystem 1023

Auxiliary pump 1023 a

Check Valve 1023 b-Prevents back pressure on the auxiliary pump.

A rotary valve selects among reagent and waste bottles, or output to thecell. The syringe pump aspirates or dispenses to the selected valve.

Either by hand or using laboratory automation, the user places a 96 wellmicro-titre plate on the platform.

An actuator moves the platform with the plate into position.

Transfer beads into cell

-   -   a) Probe assemblies descend to near top of well    -   b) Slow compression of the puffer tubing to displace fluid into        the wells    -   c) Probe assemblies move to bottom of well    -   d) Rapid release of puffer tubing to draw fluid and beads into        the bead tubes

FIG. 119 sets forth the the governing design principles.

Description of the 6 Steps

The 6 steps are described in detail, as follows:

1) The Prime Cell Step

FIGS. 122 a-e show the basic sequence of the prime cell step.

The purpose of the Prime Cell step is to configure the cell and itsassociated fluidic components in a state that allows effective transferof beads from the well plate 1012 to the cell. Such a state ischaracterized by having the entire fluidic system, from the syringe pumpto the probe tips filled with a buffer solution and having substantiallyall the air removed from the fluidic system, including both small airbubbles and larger cavities. Once in this state, the fluidic system isconsidered “stiff” from a fluidic point of view, and is capable ofsupporting bead transfer, bead loading and bead flushing operations.

The following sequence, which relies on a syringe pump as the motive forfluids through the system, is designed to prime the cell:

Displace fluid in system with air,

Displace Air with Ethanol,

Displace Ethanol with Water, and

Displace Water with Buffer.

Each of the four basic elements of the prime cycle has a specificpurpose, as does the order of operations. The importance of the firststep, pushing air through the system to displace any fluid that mayalready be in the system, was found to help with the second step,ethanol purge; though it is still unclear why it helps. Ethanol is thefirst fluid pushed through the system after purging with air. Ethanolhas a very low surface tension and is a good wetting agent; propertiesthat make it ideal for removing bubbles throughout the system,especially in the cell where bubbles trapped in the small cross-sectiondevice are most difficult to remove. Once the interior surfaces of thefluidic system are wetted with ethanol and the air bubbles are removed,water is pushed through. Although the ethanol is highly effective atremoving air bubbles, the one source of trapped air ethanol cannotremove resides in the chimney. The pocket of air trapped in the chimneyis a consequence of pushing fluid down the chimney rather than up thechimney. The natural tendency of the air in the chimney is to rise sinceit is less dense than all the fluids. When ethanol is pushed through, itsimply spills around the air pocket as it travels from the top of thechimney to the bottom on its way to the cell. The spilling effect, aresult of very low surface tension, prevents the air from beingdisplaced by the liquid. To overcome this, water is pushed through thesystem next. Because the ethanol wets all surfaces the water can comethrough next and wet the surfaces by simply displacing the ethanolrather than trying the wet dry surfaces directly. Once the waterdisplaces the ethanol, its high surface tension allows it to form ameniscus at the top of the chimney, which follows the shape of thechimney as it travel from the inlet at the top of the chimney to thecell at the bottom. Provided the inside diameter of the chimney neverexceeds a critical diameter (approximately 9 mm for a round geometry andless for shapes that deviate from round), it is possible to support acolumn of water above the air without spilling around the air pocket. Asthe water is introduced into the chimney by the syringe pump the pocketof air is continuously pushed down toward the bottom of the chimney andeventually out through the cell and finally through the probe tips. Inaddition to the critical diameter, it is also important that surfacesand transitions inside the chimney be smooth and continuous; asperitieswill tend to break the meniscus as it travels slowly down the chimney.Once the water is pushed entirely through the system and the cell isfree of all air both in the form of bubbles and cavities the sequenceproceeds to the final step, which is to simply displace the water with abuffer solution. The system is now considered primed.

2) The Transfer Beads to Cell Step

FIGS. 123 a-e show the basic sequence of the step for transferring ofbeads 8 to the cell or well-plate 1008.

The transfer process refers to the movement of beads from the well-plate1012 to the cell 1008 through a path, which includes the transfer tube,the cell and finally the chimney. In most cases, beads begin theirjourney at the bottom of a round bottom well; since they are denser thanthe buffer fluid they sink to the bottom of the well. The method oftransferring beads from the well plate involves vacuuming them off thebottom of the well with an open-ended tube attached to the cell, calleda transfer tube. At the distal end of the transfer tube is the probe tipwhich has attached to it a cone-shaped vestige designed to enhance theflow rate around beads that are more than a few tube diameters away fromthe center of the tube, thereby enhancing the efficiency of the transferprocess. However, unlike a typical vacuum whereby the flow rate isalways in the same direction (i.e. into the vacuum) the method employedhere involves alternating the direction of the flow and varying the rateof flow.

Responsibility for this action is a device called a “puffer,” whichconsists of a flexible silicone tube approximately 2″ long by ¼″diameter. The tube is connected in-line between the syringe pump and thecell and is placed between two metal surfaces, on of which moves inorder to squeezed the tube. Fluid rushes out of the tube when it'ssqueezed and back in when it's released. Since one end of the tube isdead-ended at the syringe and the other end (the part that goes into thewell with the beads) is open, the net flow is always through the openend, both inward and outward.

Beads are transported from the well-plate to the cell by repeating acycle of slow contractions and fast expansions of the puffer tube. Slowcontractions re-set the puffer tube to a state whereby a vacuum can beapplied to the transfer tube (expansion), thereby pulling beads towardthe cell. While the flow rate is slow liquid moves past the beadswithout carrying them very far in the direction of the flow. While theflow is rapid, beads are effectively moved in the direction of the flow.Therefore, repeating the cycle causes the beads to acquire a net motionin the direction of the fast flow. This method is employed to lift beadsout of the well plate and transport them to the cell during the transferprocess and move beads out on the groove plate during the load processand remove beads from the grooves and flush them out of the cell duringthe flush process.

Another key element of the fluidic architecture with regard to thetransfer process is the chimney. The chimney is made to have a rapidlyincreasing inner diameter starting from the point at which it isattached to the cell. The purpose of the large inner diameter is todecrease the flow rate to the extent that beads cannot travel past thechimney and become lost in tubing. The inner volume of the chimney isdesigned to be 2 to 5 times larger than the volume of fluid displaced bythe puffer during the transfer process. It was found that beads enteringthe chimney at high rates of speed travel about ¾ the height of thechimney before the flow is reversed (slow contraction) which then pushesthe beads to the bottom of the chimney and even out onto the grooveplate. After a certain number of puffer cycles (10-15), the puffer stopsand beads fall under their own weight to the bottom of the chimney andpile up in small rectangular opening called the line port. Thedistribution of beads in the port is uniform across the opening, whichis important for the next step; bead load.

Beads are considered transferred after a set number of (empiricallydetermined) puffer cycles. Once the beads are transferred excess cyclescause them to harmlessly rise and fall in the chimney. Therefore,without a means of feedback, the transfer process is always run with anexcess number of cycles to ensure that a high percentage of beads aretransferred. Efficiencies that range between 95 and 99.9% are obtainedafter about 15 cycles, approximately 40 seconds. The proc0ess concludeswith beads settling to the bottom of the chimney on the groove plate ina pile substantially uniform in distribution within the port, aconsequence of randomization caused by turbulent flow in the chimney.

The port, which is defined as the opening of the chimney to the cell, isrectangular in shape, approximately 6 mm long and 250 μm wide. It issurrounded on three edges by the gasket, which forms the perimeter ofeach of the 8 independent lanes. The fourth edge is open to the laneleading to the grooved region. The back edge of the port is aligned asclosely as possible to the edge of the gasket so as to minimize deadzones in the flow field caused by eddy currents. Gaps that range from 0to 50 μm were found to eliminate such dead regions behind the opening ofthe port where beads could potentially become stuck. Similarly, thewidth of the port opening is large enough to ensure beads don't form alog jam but small enough ensure the velocity of the flow through anyportion of the opening is sufficiently large to carry beads out of theport region during the bead load process. The range of openings found tobe effective were 200 to 400 μm. The length of the port opening, whichspans nearly the entire 7 mm width of the lane, was found to produce themost uniform distribution of beads in the grooved region of anycombination of port and gasket shapes. Other geometries tried involvedports of various sizes of circles with gaskets cut into linear tapers,horn shaped tapers and parabolic shaped taper, which depending on theflow rate, produced either narrow beam-like distributions or lobeddistributions characterized by a low density region of beads in themiddle of the lane and high density regions near the edges of the lane.Both types of profiles produced unacceptably low total packingdensities, a feature that plays heavily into the overall throughput ofthe instrument. Unlike the circular port shapes that rely on the flowfield to produce distribution functions, the rectangular port shapeallows the beads to form a uniform distribution across the width of thelane by the simple process of mixing in the chimney then settling to thebottom.

Another feature of the cell that plays in important roll in the dynamicsof bead transport is the thickness of the gasket. The gasket not onlydefines the perimeter of the lane around the grooved region and theports, it also defines the height of the column of fluid in the cell.With a density of 2.2 glass beads sink in aqueous solutions, which meanswhen they are in the cell they will lay on the surface of the grooveplate (the bottom of the cell), where the velocity of the laminar flowis close to zero. When the height of the laminar flow field (thicknessof the gasket) becomes very large compared with the diameter of the beadthe velocity of the flow intercepting the bead approaches zero.Therefore, to maximize the interaction of the bead with the flow fieldthe gasket thickness should be kept as thin as possible.

Countering this requirement are two issues that occur when the gasket istoo thin. The first pertains to the persistence of small air bubbles.The smaller the gap between the groove plate and the top plate theharder it becomes to flush small air bubbles away. It was found that agasket thickness of less than 300 μm resulted in such problems. Thesecond relates to the pressure drop across the cell during the transfercycle. Because the entire pressure generated by the puffer duringtransfer drops across both the cell and the transfer tube, since they'rein series with each other, the impedance of the cell cannot be muchlarger than the transfer line. Otherwise the flow rate at the distal endof the transfer tube will be insufficient to cause bead transport out ofthe well. Therefore it is important to balance the impedance of thetransfer tube with the cell. Again, the minimum gasket thickness wasfound to be around 300 μm.

3) Load the Beads into the Grooves

FIGS. 124 a-b show the basic sequence of the step for loading of thebeads 8 into the grooves of the well-plate 1008.

4) Scan the Beads in the Well-Plate

The step for scanning the beads in the well-plate 1008 is consistentwith that described above.

5) Flushing the Beads from the Grooves

FIGS. 125 a-b show the basic sequence of the step for flushing the beads8 from the grooves of the well-plate 1008.

FIG. 126: The Groove Plate Design

FIGS. 126 a and b show the groove plate design.

FIGS. 127 a-d: Bead Alignment Feasibility Experiments

FIGS. 127 a-d relate to bead feasibility experiments, including FIG. 127a that shows bead alignment feasibility experiments with performancerequirements and drivers and the basic parameters of the experiment;FIG. 127 b that shows multiplex ranges; FIG. 127 c that shows bead lossfeasibility experiments; and FIG. 127 d that shows bead flushfeasibility experiments.

FIGS. 128-133

FIGS. 128-133 show more detailed diagrams of components of the basicarchitecture 1001.

The Scope of the Invention

Unless otherwise specifically stated herein, the term “microbead” isused herein as a label and does not restrict any embodiment orapplication of the present invention to certain dimensions, materialsand/or geometries.

The dimensions and/or geometries for any of the embodiments describedherein are merely for illustrative purposes and, as such, any otherdimensions and/or geometries may be used if desired, depending on theapplication, size, performance, manufacturing requirements, or otherfactors, in view of the teachings herein.

It should be understood that, unless stated otherwise herein, any of thefeatures, characteristics, alternatives or modifications describedregarding a particular embodiment herein may also be applied, used, orincorporated with any other embodiment described herein. Also, thedrawings herein are not drawn to scale.

Although the invention has been described and illustrated with respectto exemplary embodiments thereof, the foregoing and various otheradditions and omissions may be made therein and thereto withoutdeparting from the spirit and scope of the present invention.

1. A method for aligning microbeads to be read by a code reading orother detection device, comprising the step of: providing microbeads toa positioning device, each having an elongated body with a code embeddedtherein along a longitudinal axis thereof; aligning the microbeads withthe positioning device so the longitudinal axis of the microbeads is ina fixed orientation relative to the code reading or other detectiondevice.
 2. A method according to claim 1, wherein the positioning deviceis a plate having a multiplicity of grooves therein.
 3. A methodaccording to claim 1, wherein the method includes agitating the plate toencourage the alignment of the microbeads in the grooves.
 4. A methodaccording to claim 1, wherein the microbeads are cylindrically shapedglass beads between 25 and 250 microns in diameter and between 100 and500 microns long.
 5. A method according to claim 1, wherein themicrobeads have a holographic code embedded in a central region thereof.6. A method according to claim 1, wherein the code is used to correlatea chemical content on each bead with a measured fluorescence signal. 7.A method according to claim 1, wherein each microbead is substantiallyaligned in relation to its pitch and yaw rotational axes.
 8. A methodaccording to claim 1, wherein the plate has a series of parallel grooveshaving one of several different shapes, including square, v-shaped orsemi-circular.
 9. A method according to claim 1, wherein the plate is anoptically transparent medium including boro-silicate glass, fused silicaor plastic, and the grooves are formed therein.
 10. A method accordingto claim 1, wherein the grooves have a depth that is dimensioned to beat least the diameter of the microbeads, including at least 110% of thediameter of the microbead.
 11. Apparatus for aligning microbeads to beread by a code reading device, comprising: a positioning device foraligning microbeads, each microbead having an elongated body with a codeembedded therein along a longitudinal axis thereof, so the longitudinalaxis of the microbeads is positioned in a fixed orientation relative tothe code reading device.
 12. Apparatus according to claim 11, whereinthe positioning device is a plate having a multiplicity of groovestherein.
 13. Apparatus according to claim 1, wherein the apparatusincludes means for agitating the plate to encourage the alignment of themicrobeads in the grooves.
 14. Apparatus according to claim 1, whereinthe microbeads are cylindrically shaped glass beads between 25 and 250microns in diameter and between 100 and 500 microns long.
 15. Apparatusfor aligning an optical identification element, comprising: the opticalidentification element having an optical substrate having at least aportion thereof with at least one diffraction grating disposed therein,the grating having at least one refractive index pitch superimposed at acommon location, the grating providing an output optical signal whenilluminated by an incident light signal, the optical output signal beingindicative of a code, and the optical identification element being anelongated object with a longitudinal axis; and an alignment device whichaligns the optical identification element such that said output opticalsignal is indicative of the code.
 16. Apparatus according to claim 15,wherein the alignment device is a plate having a multiplicity of groovestherein.
 17. Apparatus according to claim 15, wherein the plate is adisk and the multiplicity of grooves are concentric circles or a spiral.18. Apparatus according to claim 15, wherein the alignment device is atube having a bore for receiving the optical identification element.